Tomato pollen tube development and carbohydrate fluctuations in the autotrophic phase of growth

Ripe pollen has different soluble and insoluble carbohydrates in variable amounts. Pollen germination and pollen tube growth were studied in a tomato cultivar (Solanum lycopersicum L. cv. Platense) with atypical pollen among tomatoes due to its very low amount or absence of sucrose. In vitro assays were performed using a culture medium without carbohydrates to explore whether there is an autotrophic phase of pollen tube growth, and if there is, describe it, and to analyze the fluctuations of endogenous carbohydrates (soluble carbohydrates, starch, pectins, and callose). Pollen germination was fast (ca. 10 min) and a definite autotrophic phase was observed. Soluble carbohydrates and pectins showed the most substantial changes during this period, even after 10 min. A small amount of callose was observed in the ripe pollen and pollen tubes. Pectins were the most abundant pollen tube wall component. Pollen can be considered starchless; starch was not involved in the autotrophic phase of growth. Other types of substances must be connected with the carbohydrate metabolism, because the fluctuations of the different substances did not follow balanced stoichiometric relationships. Pollen germination and pollen tube elongation was sustained autotrophically, even though sucrose was absent and starch was negligible in pollen grains. The type of pollen reserves and the fast pollen tube formation could be selective advantages in this cultivar.     

Studies on Cellular Site of Calcium Action in Promoting Pollen Growth

Studies were conducted to determine cellular site of Ca action in promoting pollen growth of Crinum asiaticum and a few other species. The following experimental results have strongly indicated that Ca binding takes place in pectins of the pollen tube walls. This appeared to increase the wall rigidity and to regulate permeability of the pollen cells thereby enhancing pollen growth. Radioactive Ca incorporation was observed exclusively in the pollen tube wall regions. The promoting action of Ca on pollen growth disappeared when pectinase was supplemented to the media. This was not the case with cellulase and other enzymes used. Methyl donors promoted pollen growth, and the promotion was more than doubled if Ca ions were present. Ethionine, on the other hand, inhibited tube elongation and exhibited no Ca effect. Growth of pollen tubes in oscillaled liquid media during elongation was poorer than growth in standing media. The Ca effect was also reduced when pollen was oscillated. The observations made of the reduced rate of ⁴⁵Ca incorporation when pollen was washed in water, and the hydroxylamineferric chloride test, have indicated that a considerable portion of these pectins are cold-water soluble.     

Influence of Spectral Quality of Light on Pollen Tube Elongation in Arachis hypogaea

The effect of light of different wave lengths (red RL, blueBL, yellow YL, green GL and far-red FR) on pollen tube elongationhas been studied in Arachis hypogaea. Short exposure of pollento RL caused enhanced tube elongation and this effect was reducedby FR and BL. The effects of RL and FR were mutually reversible.Similarly, the inhibitory effect of blue light could be overcomeby IAA and riboflavin separately whereas acetylcholine and GA₃mimicked the RL effect. The activities of peroxidase (PO) andmalate dehydrogenase (MDH) were increased by RL and IAA whereasBL increased the activity of IAA-oxidase. The regulatory roleof light of different spectral composition in controlling pollentube elongation is discussed. Arachis hypogaea, pollen tube elongation, light     

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

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

The Growth Activity and Fertilizing Capacity of Submersely Cultivated Pollen Tubes of Nicotiana tabacum L.

Tobacco pollen was cultivated in shaken suspension culturesand the growth estimated by weighing the mass of germinatedpollen separated from the nutrient solution. A formula for calculatingmean pollen tube length from the weight of the culture has beenderived for this pollen species. The growth of pollen tubes in vitro is shown to have a rhythmiccharacter. The rapid growth comparable to the mean growth ratein styles is limited to short time intervals alternating withprogressively extending periods of very depressed growth, whichceased entirely after 10–12 h of cultivation. When tested by placental pollination in vitro, the fertilizingcapacity of the pollen culture was found to increase duringthe first hour of cultivation but to decrease steadily thereafter.On the other hand, with the application of pollen tubes fromculture on stigma, a short precultivation period and even themere wetting of pollen had a negative effect on the seed set.With pollen cultivated longer than 4 h, no seed formation wasobserved.     

Effects of myrmicacin (β-hydroxydecanoic acid) on protoplasmic movement and ultrastructure ofCamellia japonica pollen

Summary While tube elongation of growing pollen ofCamellia japonica was stopped by treatment with 50–100 ppm of myrmicacin, protoplasmic movement in the pollen tube still continued. However, higher concentration (200 ppm) of the inhibitor arrested the movement. The vesicles containing membrane substances disappeared at the tip of the tube of the growth-inhibited pollen. Removal of the inhibitor resulted in the reappearance of the vesicles at the tip region and tube elongation was restored.     

Polyethylene glycol improves the in vitro growth of Brassica pollen tubes without loss in germination

The culture media for the in vitro pollen assay of Brassicaspecies have so far shown good percentage germination, but limitedpollen tube growth. It is reported here that by lowering sucroseconcentration from 20% to 5 or 10 % and by adding polyethyleneglycol 4000 in the culture medium, high percentage germinationand high tube growth (10 times that in the standard media) canbe achieved in three species of cultivated Brassica. This improvedmedium should be useful in fundamental and applied studies onthe pollen biology of Brassica species. Key words: Brassica, pollen, pollen germination, pollen tube growth, polyethylene glycol     

In Vitro Phosphorylation of Proteins in IAA-Treated Mesocotyls in Zea mays

Five-mm sections of elongation zones of Zea mesocotyls wereincubated for designated periods with various concentrationsof IAA. In vitro protein phosphorylation in the soluble fraction(85,000 x g supernatant) prepared from the sections was analyzedby sodium dodecyl sulfate-polyacrylamide gel electrophoresis.The phosphorylation of proteins in the soluble fraction thathad been prepared from sections incubated for 20 min in thepresence of 10{small tilde}^s M IAA was greater than that inthe sections incubated for 20 min without IAA. The amount ofphosphorylation of proteins per protein became higher when higherconcentrations increased (10{small tilde}⁸—10{small tilde}⁵M).The growth of sections incubated in the presence of 10{smalltilde}⁸ M IAA or higher concentrations was greater than thatof sections incubated in the absence of IAA. The promotion ofgrowth by IAA was greater at higher concentrations of IAA. Proteinsin the soluble fraction, prepared from sections incubated for20 min in the presence of 10{small tilde}⁵ M IAA, were phosphorylatedin the presence of either 10 fM cAMP, 10 µM cGMP, 100µM W-7, 100 µM W-5, 20 µM H-7 or 20 µMHA1004. The calmodulin antagonist, W-7, and the inhibitor ofprotein kinase C, H-7, inhibited the phosphorylation of proteinsstimulated by incubation with IAA. These results suggest thatIAA promotes cell elongation via protein phosphorylation thatdepends on calmodulin-dependent protein kinase and protein kinaseC. (Received November 29, 1995; Accepted May 20, 1996)     

Pistil Exudate Production and Pollen Tube Growth in Lilium longiflorum Thunb.

Exudate production in the pistil of Lilium longiflorum was studiedin relation to pollen tube growth, using scanning electron microscopy(SEM), transmission electron microscopy and light microscopy.In contrast with conventional fixation for SEM, during whichthe exudate of L. longiflorum largely washes away, the exudateremains present through freezing in case of cryo-SEM. Usingthe latter method we observed that exudate production on thestigma and in the style started before anthesis. Just underneaththe stigma the exudate was first accumulated at the top of eachsecretory cell, followed by a merging of those accumulationsas exudate production proceeded. Exudate is also produced bythe placenta. It was however not possible to determine whetherany of this fluid originated from the micropyle. Apart fromthe cell shape and the cuticle present in between the secretorycells, the ultrastructure of the secretory cells covering theplacenta was comparable to those of the stylar canal. The transferwall of the secretory cells of the placenta originated fromfusing Golgi vesicles but the endoplasmic reticulum seemed tohave an important role as well. After pollination the pollen tubes grew across the stigma andentered the style through one of the slits in the three stigmalobes. The pollen tubes grew straight downward through the styleand were covered by exudate. As the pollen tubes approachedthe ovary their growth was restricted to the areas with secretorycells. In the cavity the pollen tubes formed a bundle and theybent from this bundle in between the ovules towards the micropylarside. There they bent again to stay close to the secretory cells.After bud pollination the pollen tube growth was retarded. Laterarriving pollen tubes had a tendency to grow close to the secretorycells of the style, which resulted in a growth between thesecells and preceding pollen tubes. If there was still a littleexudate produced, it resulted in a lifting up of the pollentubes, out of the exudate. The relationship between exudateproduction and pollen tube growth is discussed. Both the speedand the guidance of the pollen tube seemed determined by theproperties of the exudate.Copyright 1994, 1999 Academic Press Cryo-scanning electron microscopy, exudate, Lilium longiflorum, lily, ovary, pollination, pollen tube growth, secretory cell, stigma, style     

Effect of Water Loss on Germination Ability of Maize (Zea mays L.) Pollen

The correlation between water content and viability of maizepollen grains was studied on the basis of the germination abilityof pollen from a single cross hybrid. There was found to beclose correlation between viability of the grains and theirtolerance to desiccation. Most of the pollen grains in the hybridexamined survived a reduction by almost 50 per cent of the originalwater content without loss of normal function. With water lossgreater than this, less vigorous pollen grains died or losttheir ability to form pollen tubes. Consequently, when pollinationwas carried out using pollen with a water content reduced bymore than 50 per cent, only the most tolerant pollen grainssurvived to take part in the competition which precedes fertilization.Dry pollen grains required a longer period to establish adhesionto the stigma surface and to initiate pollen tubes than pollengrains with higher water content, but otherwise their behaviourwas normal. If more than 80 per cent of the original water contentwas lost, disturbances occurred in the physiology of the grainssurviving the treatment. This was exhibited as death or a reductionin rate of pollen tube growth. Drying of pollen by an amount which does not irreversibly damagethe more tolerant grains could possibly be used as a means ofpollen selection. Zea mays L., maize, pollen viability, pollen treatment, dehydration, pollen tube     

The Importance of Light Intensity for Pollen Tube Growth and Embryo Survival in Wheat x Maize Crosses

The success of Triticum aestivumxZea mays crosses, used to producewheat doubled haploids, is influenced by light intensity. Toexamine the basis for this response, pollen tube growth, embryosurvival and indicators of photosynthetic rate were measuredin two wheat cultivars (‘Karamu’ and ‘Kotuku’)crossed with maize at two irradiance levels (250 or 750 µmolm^-2s^-1, PAR). Pollen tube growth was significantly affectedby light intensity in ‘Karamu’ plants but not in‘Kotuku’ plants, despite both cultivars being pollinatedby the same maize source. The percentage of pollen tubes reachingthe cavity between the ovarian wall and integuments, or in themicropyle of ‘Karamu’ plants at high light intensity(65%) was nearly three-times greater than that at low lightintensity (22%). Thus, either low light intensity can affectthe maternal wheat plant in a way that inhibits pollen tubegrowth and/or high light intensity may promote pollen tube growthin ‘Karamu’ plants. Significant differences in ratesof electron transport in plants grown at the two light intensitiesindicated that the rate of photosynthesis may also have an effecton pollen tube growth. These results have importance for improvingthe efficiency of wheat x maize crosses and other wide cerealcrosses. Copyright 2001 Annals of Botany Company Intergeneric hybridization, light intensity, pollen tube growth, embryo survival, Triticum aestivum, wheat,Zea mays , maize     

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.     

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     

Proline, Hydroxyproline, and Lily Pollen Tube Elongation

Cytoplasm of freshly-harvested, ungerminated Lilium longiflorum,cv. Ace pollen contains 0.14 per cent soluble and 0.35 per centprotein-bound proline (pro). Their metabolic fates in germinationand tube elongation are not known with certainty. Here is reportedconversion of pro to hydroxyproline (hyp)—containing constituentsas well as distribution and isolation of these constituents.Colorimetry revealed pro and hyp in wall, trichloroacetic acid(TCA)—precipitable, and TCA-soluble cytoplasmic fractions.A balance sheet summarizing quantitative changes in pro andhyp for these fractions revealed that TCA—precipitablecytoplasmic pro could be a precursor to wall-bound pro and asubstrate for hydroxylation yielding cytoplasmic and wall-boundhyp. To determine whether hyp was a component of tube and/orgrain walls, pollen was allowed to germinate 1.5 h and thentransferred to sorbitol medium which prevented further tubeelongation. Hyp was absent from walls of transferred pollen.Electron microscope autoradiography of tubes exposed to ²H-prosuggested that a pro- and/or hyp-containing constituent waslocalized in the growing tip. Light microscope autoradiographyof intact tubes labelled with ¹⁴C-pro showed that the constituentwas distributed throughout the pollen tubes. Gel filtrationof hyp-containing material enzymically released from walls supportedthe view that they contained hyp-glycopeptides.     

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.     

Differential roles of microtubule and actin-myosin cytoskeleton in the growth of Pinus pollen tubes

The behavior and role of the microtubule (MT) and actin-myosin components of the cytoskeleton during pollen tube growth in two species of Pinus were studied using anti--tubulin, rhodamine-phalloidin, anti-myosin, and the appropriate inhibitors. Within germinated pollen tubes MTs were arranged obliquely or transversely, but in elongated tubes they were arranged along the tube's long axis. MTs were localized in the tube tip region, excluding the basal part. Altered growth was found in pollen tubes treated with colchicine; the tips of many pollen tubes incubated in the liquid medium were branched and/or rounded, and those in the agar medium were divided into many branches. Both the branching and the rounding were considered to be caused by the disturbance of polarizing growth of the tube due to MT disorganization with colchicine treatment. Actin filaments (F-actin) were found in the major parts of many pollen tubes along their long axis, excluding the tip region. In a few tubes, however, F-actin was distributed throughout the tube. The areas in the pollen tube containing F-actin were filled with abundant cytoplasmic granules, but the areas without F-actin had very few granules. The tube nucleus, which migrated from the grain area into the tube, was closely associated with F-actin. Germination of pollen grains treated with cytochalasin B was little affected, but further tube elongation was inhibited. Myosin was identified on cytoplasmic granules and to a lesser extent on the tube nucleus in the pollen tubes. Several granules were attached to the nuclear envelope. Tube growth was completely inhibited by N-ethylmaleimide treatment. In generative cells that were retained in the pollen grain, both MT and F-actin networks were observed. Myosin was localized on the cytoplasmic granules but not on the cell surface. In conclusion, it was shown that actin-myosin and MTs were present in gymnospermous Pinus pollen tubes and it is suggested that the former contributed to outgrowth of the tubes and the latter contributed to polarized growth. Several differences in the behavior of cytoskeletal elements in generative cells compared to angiosperms were revealed and are discussed.     

Influence of Temperature and Ageing of Ovules and Pollen on Reproductive Success in Trifolium repens L.

Four clones of Trifolium repens L., originating from Iceland,Denmark and Israel, were cultivated in controlled environmentchambers at 10, 14, 18 and 22°C. Seed set at 10°C was,on average, 45% lower than at 18°C. Generally, seed frequencieswere higher in the stylar ovule positions than in the basal.This polarization became more pronounced at low temperatures.At least 30% of the decrease in seed set was caused by inadequatepollen tube growth. This was further supported by in vitro studieswhich showed that pollen developed significantly shorter pollentubes at low temperatures and a strong positive correlationbetween the capacity of male parents to induce seed set in basalovule positions and the length of pollen tubes in vitro. Femaleage, determined by days relative to anthesis, was also foundto have a significant influence on seed set, i.e. when pollinationwas delayed until the fifth day of flowering, seed set decreasedby an average of 60% when compared to florets pollinated onthe first day of anthesis. Pollen age did not influence seedset. Ageing of pollen decreased in vitro germinability, tubeelongation and the percentage of pollen tubes with sperm cells.Seed set, pollen germinability, pollen tube lengths, and abilityto form sperm cells in vitro varied significantly among clones.Copyright1994, 1999 Academic Press Trifolium repens, white clover, temperature, pollen tube, sperm cells, ovule fertility, seed set     

Pollen Diffusates of Crotalaria retusa and their Role in pH Regulation

Details of the release of proteins and amino acids from culturedpollen grains and the role of the leached metabolites in pollengermination, pollen tube growth and regulation of pH of theculture medium in Crotalaria retusa have been investigated.In unbuffered media, satisfactory pollen germination and tubegrowth occurred over a wide range of pH values 4.0–9.0.This was related to the ability of pollen diffusates to shiftthe pH to 6.25 in all these media. Similar pollen germinationand pH shift was observed when the pollen was eluted twice beforeculturing. When the pH shift was reduced by using buffered media,optimal germination and tube growth occurred only at pH 6.0.Pollen diffusates had a strong buffering capacity. Proteinsand amino acids released from pollen do not seem to have a directrole in pH regulation. The components involved in pH regulationmay originate from the pollen wall as well as from the cytoplasm. Crotalaria retusa L, pH regulation, pollen diffusates, pollen germination     

High temperature limits in vivo pollen tube growth rates by altering diurnal carbohydrate balance in field-grown Gossypium hirsutum pistils

It has recently been reported that high temperature slows in vivo pollen tube growth rates in Gossypium hirsutum pistils under field conditions. Although numerous physical and biochemical pollen-pistil interactions are necessary for in vivo pollen tube growth to occur, studies investigating the influence of heat-induced changes in pistil biochemistry on in vivo pollen tube growth rates are lacking. We hypothesized that high temperature would alter diurnal pistil biochemistry and that pollen tube growth rates would be dependent upon the soluble carbohydrate content of the pistil during pollen tube growth. G. hirsutum seeds were sown on different dates to obtain flowers exposed to contrasting ambient temperatures but at the same developmental stage. Diurnal pistil measurements included carbohydrate balance, glutathione reductase (GR; EC 1.8.1.7), soluble protein, superoxide dismutase (SOD; EC 1.15.1.1), NADPH oxidase (NOX; EC 1.6.3.1), adenosine triphosphate (ATP), and water-soluble calcium. Soluble carbohydrate levels in cotton pistils were as much as 67.5% lower under high temperature conditions (34.6 °C maximum air temperature; August 4, 2009) than under cooler conditions (29.9 °C maximum air temperature; August 14, 2009). Regression analysis revealed that pollen tube growth rates were highly correlated with the soluble carbohydrate content of the pistil during pollen tube growth (r² = 0.932). Higher ambient temperature conditions on August 4 increased GR activity in the pistil only during periods not associated with in vivo pollen tube growth; pistil protein content declined earlier in the day under high temperatures; SOD and NOX were unaffected by either sample date or time of day; pistil ATP and water soluble calcium were unaffected by the warmer temperatures. We conclude that moderate heat stress significantly alters diurnal carbohydrate balance in the pistil and suggest that pollen tube growth rate through the style may be limited by soluble carbohydrate supply in the pistil.