Profiling and functional classification of esterases in olive (Olea europaea) pollen during germination

Background and Aims

A pollen grain contains a number of esterases, many of which are released upon contact with the stigma surface. However, the identity and function of most of these esterases remain unknown. In this work, esterases from olive pollen during its germination were identifided and functionally characterized.

Methods

The esterolytic capacity of olive (Olea europaea) pollen was examined using in vitro and in-gel enzymatic assays with different enzyme substrates. The functional analysis of pollen esterases was achieved by inhibition assays by using specific inhibitors. The cellular localization of esterase activities was performed using histochemical methods.

Key Results

Olive pollen showed high levels of non-specific esterase activity, which remained steady after hydration and germination. Up to 20 esterolytic bands were identified on polyacrylamide gels. All the inhibitors decreased pollen germinability, but only diisopropyl fluorophosphate (DIFP) hampered pollen tube growth. Non-specific esterase activity is localized on the surface of oil bodies (OBs) and small vesicles, in the pollen intine and in the callose layer of the pollen tube wall. Acetylcholinesterase (AChE) activity was mostly observed in the apertures, exine and pollen coat, and attached to the pollen tube wall surface and to small cytoplasmic vesicles.

Conclusions

In this work, for the first time a systematic functional characterization of esterase enzymes in pollen from a plant species with wet stigma has been carried out. Olive pollen esterases belong to four different functional groups: carboxylesterases, acetylesterases, AChEs and lipases. The cellular localization of esterase activity indicates that the intine is a putative storage site for esterolytic enzymes in olive pollen. Based on inhibition assays and cellular localization of enzymatic activities, it can be concluded that these enzymes are likely to be involved in pollen germination, and pollen tube growth and penetration of the stigma.     

Esterases in pollen and stigma of Brassica

The dry type stigma of Brassica is covered with a continuous layer of cuticle. Cutinase and non-specific esterases may be involved in breakdown of this cuticle barrier during pollen-stigma interaction, but only a little is known about their nature and characteristics. We report here the presence of two distinct esterases from stigma and pollen of Brassica. A 33 kD esterase assayed using MU-butyrate substrate shows high activity in stigma papillae. A similar esterase from Tropaeolum pollen has been shown to possess active cutinase activity. The esterase activity in anther tissue is due to a 24 kD enzyme with substrate specificity toward acetate esters. Both enzymes require sulfhydryl groups for their catalytic activity. Immunogold labelling of antibodies raised against these esterases localised the proteins at the subcellular level. Antibodies for MU-butyrate hydrolase gave a positive signal in the cell walls of mature stigma papillae and in the tapetum and microspores during early stages of anther development. In the mature anther, a positive signal in the cytoplasm of pollen grains with some detectable localisation in the exine layer of the pollen wall was obtained. Similar results were obtained with acetate hydrolase antibodies. These esterases are thus spatially and temporally regulated in stigma and anther tissues.Abbreviations MU methyl umbelliferyl - pAbC anti-butyrate hydrolase polyclonal antibodies - pAbE anti-acetate hydrolase polyclonal antibodies     

Production of a Novel Extracellular Cutinase by the Pollen and the Chemical Composition and Ultrastructure of the Stigma Cuticle of Nasturtium (Tropaeolum majus)

Germinating nasturtium pollen (Tropaeolum majus) is shown to excrete an enzyme(s) which hydrolyzes all types of monomers from biosynthetically labeled cutin and p-nitrophenyl esters, which are model substrates for fungal cutinases. The pollen cutinase showed an optimum pH near 6.5 and was inhibited by thiol-directed reagents such as p-hydroxymercuribenzoate and N-ethyl maleimide but not by diisopropyl-fluorophosphate, an “active serine”-directed reagent indicating that the pollen enzyme is an “-SH cutinase” unlike the fungal enzyme which is a serine cutinase. Excretion of the pollen cutinase into the extracellular fluid was complete within 4 to 6 hours at 30 C. Since actinomycin D and cycloheximide showed little effect on the level of cutinase excreted, it appears that cutinase is an enzyme synthesized prior to germination. Release of cutinase into the medium did not require germination. Electron microscopy revealed the presence of a continuous cutin layer on mature stigma with extensive folds, which are proposed to play a role similar to that played by the cellular papillae found in the stigma of other plants. Chemical analysis of stigma cutin by depolymerization and combined gas-liquid chromatography and mass spectrometry showed that this cutin consists of mainly the C₁₆ family of acids. The major (70%) components were dihydroxy C₁₆ acids which consisted of 10,16- (64%), 9,16- (16%), 8,16- (12%), and 7,16- (8%) dihydroxy plamitic acid. Deuterium-labeling studies showed the presence of 16-oxo-9-hydroxy C₁₆ acid and 16-oxo-10-hydroxy C₁₆ acid in this cutin. The biochemical and ultrastructural studies indicate that the pollen tube may gain entry into stigma using cutinase excreted by the pollen.     

Stigma-surface Esterase Activity and Stigma Receptivity in Some Taxa Characterized by Wet Stigmas

Stigma-surface esterase activity and stigma receptivity througha sequence of developmental stages of the pistil have been studiedin four taxa characterized by having wet stigmas — Petuniahybrida, Nicotiana tabacum, Crinum defixum and Amaryllis vittata.The style is solid in the first two and hollow in the lattertwo taxa. In all the taxa, stigma—surface esterase couldbe detected in a thin surface layer (pellicle) from a very earlystage of pistil development, irrespective of the presence orabsence of the exudate. However, the taxa showed variation instigma receptivity. In Petunia and Nicotiana, stigmas from pistilsof all the stages supported pollen germination and tube growth.In Amaryllis and Crinum, stigmas of only the mature pistils,when the exudate is present on the stigma, supported normalpollen germination and tube growth. It is inferred that in taxacharacterized by a wet stigma and solid style, the factors requiredfor pollen germination are present from an early stage of pistildevelopment and the exudate per se is not involved in pollengermination. In taxa characterized by a wet stigma and hollowstyle, however, the pellicle does not carry the factors requiredfor pollen germination and tube growth; they appear to be presentin the exudate. Petunia hybrida Vilm, Nicotiana tabacum L., Crinum defixum, Ker-Gawl, Amaryllis vittata Ait., tobacco, pollination, pollen germination, stigmatic exudate, stigma receptivity, stigma-surface esterase, esterase activity     

The localization of nonspecific esterase and cholinesterase activity in germinating pollen and in pollen tube ofVicia faba L. The effect of actinomycin D and cycloheximide

Two types of esterases, nonspecific esterase and cholin esterase have been distinguished in germinating pollen grains and in pollen tubes ofVicia faba using cytochemical methods. The localization of each of them was different. In the hydrated non-germinating pollen grain the nonspecific esterase was present in the cytoplasm and in the intine. The cholinesterase was localized mainly in the sexine and on the outside of the plasma membrane. A particularly large agglomeration of this enzyme was found in the aperture. During germination both types of extracellular esterases were released into the medium. In the pollen tube the activity of the enzymes studied was connected with the wall of its tip. The localization of both types of esterases in the germinating pollen grain and the growing tube were independent of actinomycin D. Cycloheximid prevented the cholinesterase localization at the pollen tube tip.     

Heterostyly inPrimula. 2. Sites of pollen inhibition,and effects of pistil constituents on compatible and incompatible pollen-tube growth

Summary In incompatible (intramorph) pollinations of the heterostylousPrimula vulgaris, pollen germination or tube growth may be partially inhibited in several sites associated with the stigma or style. Blockage may occur, a) on the stigma surface through the failure of germination or of pollen tube penetration after germination, b) in the stigma head during the passage of the tube through the specialized transmitting tissue of the head, or c) in the transmitting tract of the style. None of the barriers is complete, and the prohibition of selfing or intramorph crossing depends upon the cumulative screening effect of one following upon the other. In both morphs, the germination of incompatible pollen on the stigma is enhanced in high ambient relative humidity, but many tubes still fail to penetrate the stigma. Those that do are retarded or blocked in their growth in the transmitting tissues of the stigma head and style. Crude extracts from the tissues of the stigma head and style show some differential effect on the growth of pollen tubesin vitro, and dialysates of extracts containing high molecular weight fractions show a consistent differential effect, those from thrum tissues retarding thrum tubes while having a lesser effect on pin tubes, and those from pin tissues retarding pin tubes while having lesser effect on thrum. It is suggested that the factors influencing tube growth are present in the intercellular secretions of the transmitting tract.     

POLLEN TUBE DISTRIBUTIONS IN NICOTIANA GLAUCA: EVIDENCE FOR DENSITY DEPENDENT GROWTH

I hand sectioned styles of Nicotiana glauca at intervals along their length and counted the number of pollen tubes in each section using fluorescence microscopy. Evidence of density dependent growth was found for three stages of pollen growth. Pollen germination on the stigma increased with increasing pollen population size. Pollen tube penetration in the stigma was unaffected by increasing density from low to moderate levels but was reduced at high densities. Pollen tube penetration in the style was enhanced by increasing density. This enhanced growth in the style was apparently confounded by interference among pollen tubes growing at high densities. In particular, the area of tissue able to support pollen tube growth decreases from the stigma into the lower style, which could cause overcrowding of pollen tubes growing at high densities. Enhanced pollen tube penetration with increasing density combined with interference among pollen tubes growing at high densities resulted in greater mean pollen tube lengths for populations with moderate densities. The shift from density independent growth in the stigma to positively density dependent growth in the style may represent a shift from autotrophic to heterotrophic growth stages of pollen.     

Effects of pollen competitive environment on pollen performance in Mirabilis jalapa (Nyctaginaceae)

 We examined the influence of pollen competitive environment on pollen performance in Mirabilis jalapa. We used the number of pollen grains and the number of pollen tubes per pistil as measures of pollen competition. Pollen germination, pollen tube penetration into the style, and pollen tube growth rates were used as measures of pollen performance. All three measures of pollen performance were affected by the competitive environment. Pollen germination was greatest at intermediate pollen load sizes. The percentage of germinated pollen grains that penetrated the stigma and grew into the style decreased with pollen load size. Pollen tube growth rate in the style was greater and more variable with larger numbers of pollen tubes in the style. Controlling for the degree of selection at the stigma indicated that pollen-pollen or pollen-style interactions were the likely causes of increased growth rates. Received: 28 October 1996 / Revision accepted: 24 January 1997     

Flavanoids in Pollen and Stigma of Brassica oleracea and Their Effects on Pollen Germination In Vitro

Brassica oleracea pollen was applied to a basic medium of 1.5per cent agar and 15 per cent sucrose to which flavanoids wereadded at three concentrations. Two types of agar were used;with agar 1, quercetin at a concentration of 0.5 x 10^–3per cent gave an increase in percentage germinating grains.With agar 2, an increase in germination occurred with kaempferoland naringin at concentrations of 0.5 x 10^–3 and 0.5 x10^–1 per cent respectively. Increase in pollen tube lengthoccurred with agar 2 and quercetin at a concentration of 0.5x 10^–3 per cent. The stigma tissue of B. oleracea contains at least three andthe pollen at least one glycoside of quercetin. The sugars inthe glycosides were not identified. Pollen germination and pollentube extension were not stimulated exclusively by the flavanoidspresent in the stigma. The flavanoid composition of the stigmadid not vary amongst five different S-allele genotypes, indicatingthat flavanoids are probably not directly involved in the incompatibilityreaction of B. oleracea.     

Localization of adenylate cyclase activity in Populus: its relation to pollen-pistil recognition and incompatibility

Summary Adenylate cyclase has been localized cytochemically in female and male parents as well as during the pollen-stigma interaction with an original technique employing strontium as the capture ion and adenyl imidodiphosphate as the specific substrate. The specificity of the reaction was checked by using several controls. No final specific reaction product was detected in unpollinated P. deltoides stigmas or in the P. deltoides or P. alba pollen grains used for compatible and incompatible pollinations. In the compatible cross between P. deltoides × P. deltoides, fine dense precipitates were observed in the dictyosomes and the plasma membrane and exterior to the exine of hydrated pollen grains adhering to the stigma surface. Labeling of the stigmatic pellicle was also observed after pollen adhesion and hydration. This was accompanied by a strong reactivity of the cell wall and plasmalemma of the stigma papillae at the sites of pollen tube germination on the stigma surface and at the sites of penetration of pollen tubes between adjacent papillae. In the incompatible cross between P. deltoides x P. alba, adenylate cyclase activity was still present but reduced at the stigma surface following adhesion, hydration, and germination of P. alba pollen. This activity was completely abolished after the penetration of pollen tubes between stigma papillae. These findings suggest that in Populus, adenylate cyclase activity is correlated to pollen adhesion, hydration, and germination at the stigma surface, and that the abolition of this enzyme activity could be one of the cellular events governing the gametophytic phenotype of incompatibility in the cross between P. deltoides and P. alba.     

Pectin distribution pattern in the apertural intine of Euphorbia peplus L. (Euphorbiaceae) pollen

The apertural inner layer (intine) of Euphorbia L. pollen grains has a characteristic but original structure that has paired thickenings, one on either side of the colpus. To determine the nature and role of this intine layer, pollen grains of Euphorbia peplus L. were germinated in vivo and in vitro. The germination process involves wall changes that facilitate formation of the pollen tube and its subsequent growth. In the thickenings of the intine of E. peplus, the unesterified pectin epitopes are more densely localised in the inner part of the middle intine. No such epitopes are located in the intine portion adjacent to the plasma membrane (cellulosic endintine). Unesterified pectin epitopes are also localised in the outer part of the intine but are restricted to the centre of the aperture, around and in the pore. The de-esterification of pectins is very advanced at the time of dehiscence and pollen germination. The stratification of the aperture intine may take the following pathway at the time of germination: the thin outer zone of the intine in the pore region becomes disorganised and undergoes dissolution with liberation of unesterified and esterified pectins; the middle intine thickenings undergo an important elastic modification, but without liberation of unesterified pectins; the cellulosic inner intine is the progenitor of the pollen tube wall. This special intine of E. peplus is an adaptation to the hydration process preceding germination, increasing intine and pollen grain wall elasticity.     

Computer-assisted Video Image Analysis of Spatial Variations in Membrane-associated Ca2+ and Calmodulin during Pollen Hydration, Germination and Tip Growth in Nicotiana tabacum L.

Video images of the distributional pattern of membrane-associatedcalcium (Ca²⁺) and calmodulin (CaM) have been documented andanalysed during pollen hydration, germination and tip growthin Nicotiana tabacum. Digitization of fluorescence microscopeimages of chlorotetracycline (CTC) and fluphenazine (FPZ)-fluorescenceemissions reveal that there is a maximum concentration of membrane-associatedCa²⁺ and also CaM in the vicinity of germination apertures ofhydrated pollen. With the onset of germination relatively higheramounts of Ca²⁺ and CaM were found to regionalize towards theaperture through which the pollen tube would emerge Both shortand long growing pollen tubes manifest tip-to-base Ca²⁺ andCaM gradients which are disturbed in non-growing tubes. Tubegrowth and the Ca²⁺-gradient were significantly affected byvanadate and verapamil suggesting that both a vanadate-sensitiveCa²⁺-transport system and verapamil-sensitive Ca²⁺ channelsare involved in maintaining Ca²⁺ homeostasis during pollen germinationand tube growth. The possible interactions of Ca²⁺ and CaM withdifferent cytoskeletal proteins modulating organelle movementare also briefly discussed. Image analysis, calcium, calmodulin, Nicotiana tabacum L., pollen germination, pollen tube, tip growth, Ca²⁺-channels, Ca²⁺ transport ATPase     

Papillate Tepal Hoods and Delayed Pollen Germination inAlbucaL. (Liliaceae)

In the genusAlbuca, pollen germination is delayed until thetepals close around the stigma immediately before the onsetof floral senescence. At this time, papillae on the tepal apicesand the stigma swell and produce an exudate in which pollenrapidly germinates. No pollen germination occurs when the tepalsare artificially prevented from closing around the stigma.Copyright1998 Annals of Botany Company Albuca, Liliaceae, delayed pollen germination, gametophytic self-incompatibility, perianth contributing to pollen germination, pollen tube inhibition in ovary.     

The effect of temperature on stigmatic receptivity in sweet cherry (Prunus avium L.)

Plant reproduction is highly vulnerable to environmental conditions such as temperature and, consequently, planet warming may have significant consequences on the reproductive phase with serious implication in agricultural crops. Although pollen tube growth is clearly affected by temperature, little information is available on its effect on the female side and on flower receptivity. In this work, the effect of temperature has been evaluated on stigmatic receptivity of sweet cherry in vivo, in the laboratory, and in planta, in the field. Results herein show that temperature has a clear effect on the duration of stigmatic receptivity. Thus, whereas high temperature reduced stigmatic receptivity, low temperature enlarged it. The stigma lost the capacity to offer support first for pollen penetration, second for pollen germination and, finally, for pollen adhesion. The effect of temperature was more pronounced on pollen germination and penetration than on pollen adhesion. High temperature reduced the germination capacity of the pollen as early as the first day after anthesis, a time when no apparent signs of stigma degeneration are apparent. This clear effect of temperature on stigmatic receptivity and pollen performance may have clear implication in crop performance and in establishing screening criteria of best‐adapted genotypes.     

Protein synthesis in tobacco pollen tubes: preferential synthesis of cell-wall 69-kDa and 66-kDa glycoproteins

One- and two-dimensional electrophoresis of Nicotiana tabacum pollen and pollen tube proteins confirmed that a new protein is preferentially synthesized during pollen germination and tube growth and becomes the most abundant protein in pollen tubes. Analysis of proteins extracted with sodium dodecyl sulfate (SDS) from different pollen tube fractions showed that it is the most abundant non-covalently bound wall protein, characterized by molecular mass of 69 kDa, pI between 7.9 and 8.2, and glycosylation with glucose and/or mannose. Amino acid analysis revealed relative abundance of serine, glutamic acid and glycine, but did not show the presence of hydroxyproline. According to all these characteristics, it cannot be classified as an extensin-like protein. Another prominent wall-bound glycoprotein has a molecular mass of 66 kDa and the same pI as the 69 kDa glycoprotein. These two glycoproteins are similar also in ConA binding, rate of synthesis, and rapid incorporation into pollen tube walls. Their synthesis is strongly reduced by tunicamycin and this inhibition results in the occurrence of new polypeptides in the range of 57–61 kDa. Tunicamycin also inhibited pollen tube growth. At 10 ng ml^-1 and 50 ng ml^-1 the inhibitor reduced pollen tube mass after 24 h of culture by 30% and 85%, respectively. This indicates that tobacco pollen presents a system highly sensitive to tunicamycin and that cotranslational N-linked glycosylation on the rough endoplasmic reticulum is required for 66 and 69 kDa glycoprotein formation and for pollen tube growth. Although other proteins appear during pollen germination and tube growth, the new proteins occur at low levels and seem to originate through modifications of preexisting polypeptides. In contrast to 69 and 66 kDa proteins, most proteins detected by [¹⁴C]amino acid incorporation and fluorography of gels were not revealed by Coomassie blue staining.     

Aluminium Effects on Pollen Germination and Tube Growth of Chamelaucium uncinatum. A Comparison with Other Ca2+Antagonists

An interaction between aluminium (Al) and calcium (Ca) may bea cause of Al toxicity in plants. The pollen tube is a suitablesystem to test the interaction between Al and Ca since Ca ionsplay a pivotal role in pollen germination and tube growth. Weinvestigated how Al and other known blockers of Ca²⁺-permeablechannels (trivalent cations, ruthenium red, verapamil and nifedipine)influence pollen of an Australian native species Geraldton waxflower(Chamelaucium uncinatum). Pollen germination was inhibited bymicromolar concentrations of trivalent cations (La³⁺>Al³⁺>Gd³⁺)and ruthenium red, but it was relatively insensitive to a micromolarconcentration of verapamil. Exposure of the growing pollen tubesto micromolar concentrations of Al³⁺and La³⁺, and a millimolarconcentration of Ca²⁺chelator ethyleneglycol-bis(ß-aminoethylether)-N,N'-tetraacetic acid (EGTA) led to rapid tip bursting.In contrast, exposure to Gd³⁺, nifedipine, ruthenium red, verapamiland the organic trivalent cation tris (ethylenediamine)cobalt(TEC³⁺) caused only inhibition of pollen tube growth. The Al³⁺-relatedpollen tube bursting was reduced significantly by increasingeither solution pH from 4.5 to 6 or activity of Ca²⁺from 0.25to 5 m M. In contrast, La³⁺-related pollen tube bursting wasinsensitive to changes in Ca²⁺activity. The results are discussedin terms of Al interactions with cell wall Ca²⁺and the plasmamembrane Ca²⁺-permeable channels. Copyright 1999 Annals of BotanyCompany Aluminium toxicity, Ca²⁺-channel blockers, cell wall, Chamelaucium uncinatum, pollen germination, pollen tube growth.     

Structural analysis of stigma development in relation with pollen–stigma interaction in sunflower

Structural analysis of stigma development in sunflower highlights the secretory role of papillae due to its semi-dry nature. Production of lipid-rich secretions is initiated at the staminate stage of the flowers in stigma development and increases at the receptive stage, coinciding with an extensive development of elaioplasts and endoplasmic reticulum network in the basal region of the papillae. Transfer cells, earlier identified only in the wet type of stigma, are also present in the transmitting tissue of the sunflower stigma. Attainment of physiological maturity by the stigmatic tissue, accompanying development from bud to pistillate stage, appears to affect the initial steps of pollen–stigma interaction. The nature of self-incompatibility in Helianthus has also been investigated in relation with pollen adhesion, hydration and germination. Pollen adhesion to the stigma is a rapid process in sunflower and stigma papillae exhibit greater affinity for pollen during cross pollination as compared to self-pollination. Components of the pollen coat and the pellicle on the surface of stigmatic papillae are critical for the initial phase of pollen–stigma interaction (adhesion and hydration). The lipidic components of pollen coat and the proteinaceous and lipidic components from the surface of the papillae coalesce during adhesion, leading to the movement of water from stigma to the pollen, thereby causing pollen hydration and its subsequent germination. Pollen germination (both in self-and cross-pollen) on the stigma surface and the growth of the pollen tube characterize the flexibility of self-incompatibility in sunflower. Compatible pollen grains germinate and the pollen tube penetrates the stigma surface to enter the nutrient-rich transmitting tissue. The pollen tube from incompatible pollen germination, however, fails to penetrate the stigmatic tissue and it grows parallel to the papillae. Present findings provide new insights into structural and functional relationships during stigma development and pollen–stigma interaction.     

In vitro flowering of bitter melon

Flowers were formed from shoot tips of bitter melon (Momordica charantia L.) cultured on Murashige and Skoog medium supplemented with 90 mM sucrose, 0.05 mM Fe²⁺ and 4 µM N⁶-benzyladenine (BA). The addition of 0.05 mM Fe²⁺ to the medium prevented chlorosis of the explant and promoted normal flowering. Increasing the ratio of carbon to nitrogen promoted male flower formation but intensively inhibited vegetative growth. The influence of cytokinin on the morphogenesis of the explant was highly notable. Flowers could be formed after a 15- to 20-day exposure to kinetin (Kin) or BA. Kin and BA had opposite effects with regard to the development of the explant. Kin promoted flower formation, especially female, but inhibited branch bud formation. Conversely, BA promoted branch bud formation and also promoted male flower formation when present at a concentration of 1-2 µM, but completely inhibited flower formation at 4-8 µM. Fluorescein diacetate staining and in vitro germination showed that in vitro pollen were of a fairly high viability.     

Structure and Cytochemistry of the Stigma and Pollen--Pistil Interaction in Zephyranthes

The stigma in Zephyranthes candida and Z. citrina is of thedry type with a continuous cuticle—pellicle. In some papillae,however, the terminal portion of the cuticle—pellicleis lifted upwards and occasionally even disrupted by the accumulationof a secretion product below it. Both non-specific esterasesand acid phosphatases are present on the stigma surface. Thestyle is solid with a central core of transmitting tissue whichhas conspicuous intercellular spaces containing a matrix thatincludes proteins, polysaccharides and pectic substances. Zephyranthes citrina is self-compatible while Z. candida isself-incompatible. Followng incompatible pollination in Z. candida,pollen germination is normal but pollen tube growth is inhibitedat the junction of the stigma and style. Self-incompatibilitycan be overcome by bud pollination. Protein synthesis is necessaryfor pollen germination in both species. Concanavalin A bindsto the stigma surface of both species, but does not affect pollentube penetration in Z. candida. In crosses between the two speciestypical unilateral incompatibility is observed when Z. candidais used as the pistillate parent. Zephyranthes, stigma-surface enzymes, dry stigma, pollen-pistil interaction, self-incompatibility, unilateral-incompatibility     

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