- Top of page
- Materials and Methods
- Supporting Information
In flowering plants, sexual reproduction involves the formation of the gametophyte – a highly specialized post-meiotic haploid generation, substantially reduced compared with the diploid sporophyte but dependent on it for resources. While the female gametophyte remains enveloped within the maternal sporophytic tissue, the male gametophyte – the pollen – completes only the first phase of its development protected by the sporophyte. Following programmed developmental arrest and dehydration, pollen is released from the anther and transported to the stigma. Assuming compatibility between the pollen grain and the specialized sporophytic surface of the stigma, it undergoes regulated rehydration and mobilizes stored resources (including mRNAs, complex carbohydrates and lipids) to initiate germination and highly polarized tip-based growth of the pollen tube (Taylor & Hepler, 1997). The tube acts as an altruistic ‘carrier’, transporting the sperms destined to fertilize the female gametes. In species that shed bicellular pollen, sperms are generated by the mitotic division of the generative cell during pollen tube growth, while in pollen from tricellular species, this division takes place before pollen release (Twell, 2011).
Pollen tube growth is unique to flowering plant development, with the tube tip showing exceptionally high vesicular activity and membrane synthesis, and cylindrical callosic and pecto-cellulosic walls being formed behind this focused tip region (Taylor & Hepler, 1997; Zonia, 2010). The active vesicle trafficking that supports pollen tube growth involves both exocytosis of vesicles to supply materials for extension growth, and endocytosis to recycle surplus membrane components and to import materials from the stigma. Through complex interactions with other cellular systems, turgor is a key driver of this rapid growth (Zonia, 2010; Winship et al., 2011; Zonia & Munnik, 2011; Hill et al., 2012) and, once initiated, the tube extends directionally to penetrate the sporophytic tissues of the stigmatic surface. Once within the stigmatic apoplast, the pollen tube uses signals and cues from the external cellular environment to navigate to the female gametophyte and deliver the nonmotile sperm cells to the egg and central cells (Takeuchi & Higashiyama, 2011). Pollen germination and tube growth thus emerge as highly dynamic and co-ordinated processes, integrating many different signals from the local environment to regulate growth and development.
The apparent simplicity and independence of pollen have also resulted in its use as a paradigm for plant cell growth and development (Taylor & Hepler, 1997). This, combined with the importance of pollen to scientific and applied studies of plant reproduction, has created a strong demand for systems by which pollen germination and tube growth can be induced in vitro. While some species have lent themselves to the development of effective protocols, other groups of plants remain intractable. A general correlation exists between the facility with which pollen germination and tube growth can be induced in vitro and stigma structure. Development of in vitro methods has generally proved successful in so-called ‘wet stigma’ plants, such as Nicotiana and Lilium, where the stigma produces an exudate in which pollen germinates (Allen & Hiscock, 2011). By contrast, taxa with ‘dry’ stigmas, for instance members of the Poaceae and Brassicaceae, have proved more challenging. On these surfaces, complex interactions take place that mediate highly regulated and perhaps directional uptake of water from the papillar cells of the stigmatic surface (Allen & Hiscock, 2011). It has probably been our inability to parallel these processes in vitro that has hampered successful development of protocols for efficient pollen germination and tube growth.
The model plant Arabidopsis thaliana is exceptionally practical and versatile in most aspects of experimental study, and is supported by excellent genomic and bioinformatic resources. However, its utility in studies of reproductive biology is impaired by the absence of an easy, reproducible technique for germination of pollen in vitro. Numerous papers have been published that describe methods to induce pollen germination and pollen tube growth in vitro (e.g. Li et al., 1999; Palanivelu et al., 2003; Boavida & McCormick, 2007; Bou Daher et al., 2009), and some of these have considerable utility. However, a robust and reliable method promoting high levels of germination and morphologically normal tube growth, and in which the material can be recovered for cell and molecular analysis, has remained elusive.
Here, we describe a new method that allows an unprecedented level of germination from A. thaliana pollen in vitro, followed by rapid growth of morphologically normal pollen tubes with regular callose plugs and correct localization of sperm cells. A significant innovation in our method is the use of a novel physiochemical environment that may more closely mimic the stigma, by employing a cellulose-based membrane as a support for pollen germination. Not only promoting germination and tube growth, this synthetic membrane substrate makes it easier to use the germinating gametophytes in downstream applications. Our method will permit the more effective use of A. thaliana pollen tubes in studies using microscopy (for example investigations involving the visualization of dyes and fluorescently tagged proteins), as well as research in cell biology and biophysics (such as studies of pollen tube membrane channels, transporters, wall generation and the processes of exocytosis and endocytosis) and biochemistry (for instance the extraction of RNAs and proteins from this stage of development). Finally, by subtracting from, or altering the proportion of components of the pollen germination medium, new insights are provided into the natural mechanisms that may stimulate and promote the germination and growth in planta. We anticipate that this new experimental strategy will facilitate efficient, reproducible studies of the post-hydration development of pollen from A. thaliana and its relatives.
- Top of page
- Materials and Methods
- Supporting Information
We have developed a new protocol for germinating pollen from A. thaliana in a synthetic environment independent of female reproductive tissues. The development of this protocol became a necessity as we and others have failed consistently to achieve a useful and dependable level of germination despite exhaustive trials using a range of published methods. Our protocol consistently gives germination levels > 90% with minimal variation when using higher concentrations of spermidine (0.25–0.5 mM). Further, pollen tube growth appears normal with relatively straight tubes containing callose plugs and the correct localization of the male germ unit (sperm cells) within the pollen tubes. Subsequent reduction of spermidine in the medium (to 0.1 mM) permitted the rapid formation of long tubes of normal morphology, while maintaining exceptional germination rates in vitro. Spermidine appears to promote initial germination but has an inhibitory effect on subsequent post-germination growth. Importantly, the data we present permit our protocols to be modified for use in experiments where maximal pollen germination is required, or extensive tube growth, or a compromise between the two. Although novel compounds – sulfinylated azadecalins – have been implicated as important stimulants in the germination of A. thaliana pollen (Qin et al., 2011), we have purposely restricted the composition of our germination medium to reagents readily obtained.
Key elements of the new system
The use of a cellulosic matrix
Our experiments where we have changed the components of the germination substrate components have proved informative. An integral element contributing to the performance of our system is the use of a cellulose-rich membrane layered upon the medium partially solidified by agarose. This probably results from its hydrodynamic impact, with the sheet of membrane preventing pollen grains sinking into the medium and becoming immersed in an aqueous, partially anoxic environment. While the membrane must create a ‘semi-dry’ environment better suited to development of Brassicaceae pollen which is adapted to a dry stigma, it is probable that other factors may also be responsible for the high levels of germination involved. For example, pollen may sense the physiochemical properties of this cellulosic polymer-based environment, which may more closely mimic the stigmatic papillar surface than agarose. Thus, enzymes released from the pollen wall and apoplast on hydration may use the cellulosic matrix as a substrate to generate oligosaccharides and simple sugars that could ‘feed back’ a stimulatory effect on pollen germination. Equally, this heavily processed cellulose-rich membrane itself may already contain low levels of such stimulatory compounds. Our data show that, for promoting germination, the presence of this membrane could substitute for the absence of casein enzymatic hydrolysate, myo-inositol and ferric ammonium citrate from the medium, but not spermidine. The cellulose-rich membrane also promotes extended pollen tube growth. Xyloglucans (XyGs) are known to be enriched at the tip of A. thaliana pollen tubes and the acetyl groups of XyG may promote hydrophobic interactions with the female extracellular matrix in planta (Dardelle et al., 2010). Similar interactions between extracellular matrix (ECM) components (including XyGs) and this artificial surface may stabilize the rapidly growing tip and hence promote more rapid, morphologically normal tube growth.
The inclusion of myo-inositol in the substrate medium
Myo-inositol has also been used in our growth medium, and its overlap with the cellulosic membrane in stimulating pollen growth may result from it being a precursor to UDP-glucuronic acid, itself the principal precursor to galacturonic acid, xylose, apiose and arabinose, all of which are required for cell wall biosynthesis (Kanter et al., 2005). Myo-inositol oxygenases catalyse its conversion to UDP-glucuronic acid and are encoded by a small gene family (MIOX) in A. thaliana, two of which (MIOX4 and MIOX5) are highly expressed in pollen (Kanter et al., 2005). Interestingly, experimental work suggests that uptake of exogenous myo-inositol pools is physiologically important during pollen germination and tube growth in planta (Schneider et al., 2006). Arabidopsis thaliana pollen highly expresses INOSITOL TRANSPORTER4, a plasma membrane-localized protein functioning as a high-affinity H+ symport of myo-inositol across the membrane (Schneider et al., 2006). INOSITOL TRANSPORTER4 is expressed not only in developing pollen grains but also in germinating pollen tubes in the style.
The role of spermidine in promoting pollen germination in vitro
The use of the polyamine spermidine is novel and proved very effective in stimulating pollen germination in our in vitro system. Indeed, higher concentrations ensured almost complete germination of pollen. Our observations during experiments varying spermidine concentrations suggest that part of this increase in germination may result from the ability of spermidine to eliminate local effects of pollen density, which have previously been shown to affect germination of pollen in vitro (Boavida & McCormick, 2007). In kiwifruit (Actinidia deliciosa), Falasca et al. (2010) have shown that polyamines, in particular spermidine, have a major role in normal pollen function and development, and that polyamine inhibitors in planta exerted a substantial negative effect on kiwifruit pollen germination. Loss of tomato (Lycopersicon esculentum Mill.) pollen viability in storage is correlated with degradation of transcripts involved in polyamine biosynthesis, with exogenous applications of polyamines having a restorative effect on pollen germination (Song & Tachibana, 2007).
The pivotal role of spermidine in pollen germination is more easily interpreted in the light of recent data from Wu et al. (2010), who report a role for spermidine in A. thaliana pollen tube growth, with exogenous spermidine acting to increase the cytosolic free calcium concentration in pollen tubes. Influx of calcium from the external environment is critical for pollen tube growth and a tip-localized cytosolic free calcium gradient is required for normal pollen tube elongation. Spermidine does not directly induce hyperpolarization of plasma membrane Ca2+-permeable channels, but rather spermidine oxidation by peroxisomal polyamine oxidase (PAO) generates hydrogen peroxide which acts as a second messenger to activate these channels. Interestingly, excessive extracellular spermidine concentrations activate these channels, and concomitant calcium ion influx beyond optimal levels, resulting in the inhibition of pollen tube growth. It is thus perhaps significant that our data also show that the higher concentrations of spermidine are inhibitory and there is a trade-off between stimulation of pollen germination and reduction in pollen tube growth.
The mode of action of spermidine suggests that other interacting factors are important in regulating germination and tube growth. Recently, the antioxidant glutathione has been shown to play an essential role in A. thaliana pollen germination (Zechmann et al., 2011). These findings suggest that detoxification of reactive oxygen species (such as H2O2 generated by spermidine oxidation) and redox signalling by endogenously generated glutathione may be in delicate balance, as an artificial application of high external concentrations of glutathione can inhibit pollen germination (Zechmann et al., 2011), while high external concentrations of spermidine repress pollen tube extension.
Polyamines are multifunctional molecules and spermidine may have other roles in the gametophyte. For example, work on development of the male gametophyte of Marsilea vestita, a heterosporous fern, has shown that, as in flowering plant pollen, numerous transcripts become stored and translationally inhibited during gametophyte dehydration (Boothby & Wolniak, 2011), only to be reactivated upon hydration. Importantly, spermidine plays a part in unmasking these translationally inhibited stored mRNAs (Deeb et al., 2010). It seems reasonable to speculate that spermidine plays a similar role in pollen of flowering plants in activating translation of stored transcripts when appropriate conditions for germination are encountered. Significantly, the inhibition of pollen germination in tomato by the protein synthesis inhibitor cycloheximide can be overcome by treatment with exogenous spermidine and spermine (Song et al., 2002).
Spermidine may also serve to ‘buffer’ the pollen germination in vitro against temperature variations. Boavida & McCormick (2007) report pollen germination in vitro to have a remarkably narrow optimum temperature of 22°C, a situation clearly not replicating the reproductive biology of A. thaliana in vivo. We failed to find this sensitivity to temperature using our technical approach, with low levels of pollen germination even being initiated at 37°C. The sensitivity of tomato pollen germination to high temperatures is held to result from the inhibition of S-adenosylmethionine decarboxylase, a key enzyme in spermidine synthesis (Song et al., 1999, 2002); importantly, this sensitivity could be overcome by exogenous addition of spermidine (Song et al., 1999, 2002).
The omission of spermidine from other growth media shows that germination of A. thaliana pollen, albeit at a lower level, can occur in its absence. This may be because pollen contains endogenous spermidine and related polyamines, for Handrick et al. (2010) have shown that conjugated spermidines are particularly diverse in A. thaliana pollen as a result of a complex enzymatic system. Of course, spermidine is unlikely to be the only regulator of calcium influx into the pollen cytoplasm. For example, Michard et al. (2011) have identified glutamate receptor-like proteins in A. thaliana pollen that form calcium channels regulated by exogenous D-serine supplied by the pistil. Further, in addition to providing amino acids for protein synthesis, the casein enzymatic hydrolysate may supply amino acids or small peptides which act as substitute agonists to these channels in this medium.
The role of GABA in promoting pollen tube growth
We have explored the use of GABA to increase the length of pollen tubes. GABA is a signalling molecule produced by maternal tissues to attract and guide pollen tubes to the female gametophyte (Palanivelu et al., 2003). Previous studies showed that the addition of physiological concentrations of GABA to liquid media dramatically increased pollen tube length (Palanivelu et al., 2003) and we found a similar effect when incorporated in our system. Although the molecular mechanism of the effect of GABA on pollen tubes remains to be fully elucidated, GABA appears to bind to a receptor in the cell membrane and regulates downstream Ca2+ oscillation in the cells, ultimately affecting actin organization and vesicle trafficking (Yu & Sun, 2007).
Ecotypes have different requirements for in vitro pollen germination and tube growth
Intraspecific differences exist between ecotypes of A. thaliana in their requirement for synthetic pollen germination media. Our data reveal response to pH to be an important factor distinguishing pollen from the Col-0 and Ler ecotypes in our in vitro system. The molecular basis of this pH sensitivity may result from differences in sucrose-H+ symporters between ecotypes (Sauer et al., 2004), a view supported by more recent data from Feuerstein et al. (2010), where the AtSUC1 (ARABIDOPSIS THALIANA SUCROSE-PROTON SYMPORTER 1) symporter, highly expressed in the male gametophyte and important for pollen germination and tube growth (Stadler et al., 1999; Sivitz et al., 2008), was shown to be reduced in expression in Ler compared with Col-0.
Users of our protocol wishing to apply it to other ecotypes are therefore advised to perform exploratory work to optimize media for their particular study. Our work has also underscored the importance of performing controls in parallel with test material. We found this essential for studies involving measuring pollen tube length, as, although variance within experiments is low, experiment-to-experiment differences can be considerable, even under seemingly identical experimental conditions. A major contributing factor is almost certainly batch variation in plants; for use in pollen germination studies, A. thaliana plants remain in peak condition for only a few days and differences may exist between ecotypes. For example, pollen viability of Ler has been reported to decline more rapidly than that of Col-0 (Boavida & McCormick, 2007).
We have developed an easy yet robust technology for germinating pollen from A. thaliana in vitro that employs an artificial cellulose membrane overlying an agarose layer. This system is well adapted for microscopic studies, and can also be used for rapid bioassays (for example, by incorporating toxins or signalling molecules in the medium). Many of the most interesting questions in plant reproduction and pollen tube biology – such as those involving control of pollen tube architecture, metabolic and osmotic regulation and the response to external signals – can now be explored with greater facility in this model species. The great benefits of A. thaliana as a model organism, such as the collections of mutants and its ease of transformation (for instance with constructs encoding fluorescently tagged proteins), can now be exploited fully at this unique developmental stage. The facility with which the pollen tubes may be removed from the cellulose membrane suggests that the technique will prove highly effective for rapid, clean extractions of DNA, RNA and protein from growing pollen tubes.