Pollination and stigma wounding: same response, different signal?

In Petunia hybrida flowers, both pollination and stigma woundinginduced a transient Increase in ethylene production and hastenedcorolla senescence. Ethylene production by different flowerparts was measured in situ using laser photoacoustic (LPA) spectroscopy.In pollinated flowers, ethylene was exclusively produced bythe stigma/style region whereas wounding of the stigma Inducedethylene production both by the stigma/style region and by theremaining flower parts. In aminoethoxyvinylglycine (AVG)-treatedflowers, subsequent treatment of the unwounded stigma with 1-aminocyclopropane-1-carboxylicacid (ACC) induced ethylene production exclusively by the stigma/styleregion whereas treatment of a previously wounded stigma withACC induced a simultaneous increase in ethylene production bythe stigma/style region and the remaining flower parts. Theseresults suggest that following stigma wounding, either ACC orethylene is involved in inter-organ communication. Followingpollination, the signal is apparently not directly related toethylene. In vivo ACC oxidase activity of most flower parts, includingthe gynoecium, was higher in light than in dark. Light or darkdid not influence the relative contributions of stigma/styleand remaining flower parts to the total pollination, woundingor ACC-induced ethylene production, indicating that ACC is nottranslocated. Both in excised styles and intact flowers, radiolabelledACC and its analogue -aminoisobutyric acid (AIB), applied eitherto an intact or wounded stigma, were largely immobile confirmingthat ACC is not likely to play a role in inter-organ signalling. The results collectively suggest that following stigma wounding,translocation of ethylene may be the signal responsible forinitiation of corolla senescence; following pollination thesignal is not directly related to ethylene. Key words: 1-Aminocyclopropane-1-carboxylic acid (ACC), ethylene, flower senescence, Petunia hybrida, pollination, stigma wounding     

Ethylene response to pollen tube growth in Nicotiana tabacum flowers

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

Pollination-Induced Corolla Wilting in Petunia hybrida Rapid Transfer through the Style of a Wilting-Inducing Substance

Pollination or wounding of the stigma of Petunia hybrida flowers led to the generation of a wilting factor and its transfer to the corolla within 4 hours. This was concluded from the effects of time course removal of whole styles. In this 4-hour period, pollen tubes traversed only a fraction of the total distance to the ovaries. Both pollination and wounding of the stigma immediately resulted in an increase of ethylene evolution. Accelerated wilting, however, occured only when treated styles remained connected with the ovaries, and not when they were detached and left in the flower. A wilting factor was found in eluates collected from the ovarian end of the styles, only in the case of previous pollination or wounding. In such eluates, the level of the ethylene precursor 1-amino-cyclopropane-1-carboxylic acid was below detection.

These observations suggest a material nature of the wilting factor in Petunia flowers, which rapidly passes through the style to the corolla, but which is different from 1-aminocyclopropane-1-carboxylic acid.

     

Studies on flower longevity in Digitalis

The flowers of Digitalis purpurea respond to pollination by rapid corolla abscission without any loss of corolla turgor, nor any significant loss of corolla constituents, relative to the corollas of unpollinated flowers of a similar age. The corollas of unpollinated flowers too eventually abscise, 6 d after the stigma opens, however, they do so with only a minimal loss of fresh weight or corolla constituents. Pollination causes an increase in ethylene production detectable within 1 h. Increased ethylene production occurs initially only from the upper portion of the style, later from the lower portion, and lastly, between 23 and 48 h after pollination, from the ovary plus calyx. The pollination response can be induced by exogenous ethylene, the degree of weakening of the corolla abscission zone being dependent upon the concentration and duration of the exposure period and on the stage of flower development. The regulation of ethylene biosynthesis and its involvement in the control of pollination-induced corolla abscission are discussed.     

The Control of Flower Senescence in Petunia (Petunia hybrids)

Petunia corollas wilt and abscise between one and two weeksafter detachment when maintained in distilled water in vialsat 18 °C. The onset of wilting is brought forward substantiallyby the application of 1-aminocyclopropane-1-carboxylic acid(ACC) either to the vial solution or to the stigmatic surface.Both pollination and stigma removal also shorten the time tothe onset of wilting, colour change and to abscission. In thecase of stigma removal, the life span of the corolla is shortestwhen the treatment is made at the time of flower detachment(day 0), whereas pollination has the greatest effect if it occurson day 1. Stigma damage still has an effect on corolla senescenceeven when stigma and style are removed, as long as they havebeen left in place for a few hours after treatment. Evidencefrom several experiments shows that a 17 h period is sufficientfor the full effect to be shown, and that probably there aresome effects on the corolla even if the damaged stigma is onlyleft in position for 3–6 h. Treatments which advance corolladeath (to day 3) also advance the peak of ethylene productionby the pistil (to day 1) and the corolla (to day 2). The useof silver thiosulphate (STS) overcomes all manipulative andchemical treatments used, and greatly extends vase life. Theextension occurs even when STS application is delayed for 24h, i.e. after the peak of ethylene production by the pistiland after any senescence signal has arrived at the corolla.In this case, however, the time to first morphological changeis largely unaffected, but the STS greatly extends the timeperiod between first morphological change and corolla death.The evidence suggests that early symptoms of senescence e.g.colour change and slight loss of turgor, do not automaticallylead to corolla abscission. Petunia hybrida, abscission, ACC, STS, pollination, flower senescence, ethylene     

Lack of Control by Early Pistillate Ethylene of the Accelerated Wilting of Petunia hybrida Flowers

Well before pollen tube penetration, ethylene has begun to disseminate from pollinated styles of Petunia hybrida flowers. Previous stigmatic application of aminoethoxyvinylglycine (AVG) completely prevented this ethylene synthesis, indicating that the endogenous 1-aminocyclopropane-1-carboxylic acid (ACC) in pollen is not readily converted on the stigma. Compared to other flower parts, the capacity of the ethylene forming enzyme was largest in the stigma. When applied to the stigma, ACC caused ethylene synthesis, but did not accelerate wilting, unless high concentrations (20 nanomols) were used. Upon pollination or stigma wounding, the early ethylene evolved exclusively from the gynoecium, much later followed by the synthesis of corolla ethylene. Employing wideneck Erlenmeyer flasks, the competitive inhibitor of ethylene action, norbornadiene, was applied to entire flowers in situ, with delaying effects on wound-induced wilting. In contrast, norbornadiene treatment of styles alone, using capillaries, could not postpone wilting. Pollination with foreign pollen species did not lead to accelerated corolla wilting, notwithstanding considerable synthesis of ethylene during the first 5 hours. In situ treatment of the stigma with AVG considerably delayed wound- and pollination-induced wilting. Removal of the entire AVG-treated style 6 hours after stigma wounding still allowed for the postponement of the accelerated wilting, even at very low concentrations of AVG. It is concluded that early stylar ethylene does not play a role in the acceleration of wilting but that, much later, corolla ethylene does, induced by a mobile wilting factor from the stigma, which is ACC.     

Expression of the ACC synthase and ACC oxidase coding genes after self-pollination and incongruous pollination of tobacco pistils

In tobacco, as in other species, ethylene is produced in response to pollination. Although tobacco is a self-compatible species, it displays unilateral incongruity with other Nicotianaplants. Incongruous pollination also results in ethylene production, but this production differs depending on the pollen used and is related to the extent to which pollen tubes grow in the tobacco style. In the investigation reported here we followed the expression of the ACC synthase- and ACC oxidase-coding genes upon pollination of tobacco pistils and compared self-pollination with incongruous pollination. The pattern of expression of these genes also correlated with pollen-tube growth, although wounding alone cannot explain the results obtained. We also examined the expression of these genes upon pollination of immature tobacco pistils, in which different pollen tubes grew indistinctly inside the tobacco style and reached the ovary at the same rate. In this situation no significant differences in gene expression could be observed between the different pollinations. Ethephon, a substance that produces ethylene, could, in some cases, minimize the arrest of incongruous pollen tubes inside the style.     

Pollen Tube Growth and Fertilization Efficiency in Salpiglossis sinuata: Implications for the Involvement of Chemotropic Factors

The possible involvement of chemotropic factors influencingpollen tube growth in the style and ovary of Salpiglossis wasstudied. Pollen deposited at the base of excised styles germinatedreadily, with pollen tubes penetrating the transmitting tissueand growing towards the stigma, implying that no chemotropicrequirement exists for pollen tube growth in the style. However,the position and high efficiency of seed set following limited-pollinationof intact pistils would support the involvement of localizedchemotropism operating within the ovary. Chemotropism, limited pollination, fertilization efficiency, Salpiglossis sinuata     

Pollen Presentation on Petal Combs in the Geof lorous Heath Acrotriche serrulata (Epacridaceae)

The urn-shaped flowers of Acrotriche serrulata R.Br. are unusualin two features: the mode of pollen presentation, and the corollatube which is filled with nectar at maturity, submerging thestyle and stigma. At the end of the corolla tube, the petallobes bear conspicuous subterminal hairs that usually containclumps of pollen embedded in viscous pollencoat materials. Developmentalstudies show that at anthesis, pollen from the dehisced anthersadheres to the petal hairs. When the corolla lobes reflex, thepollen is presented on the newly exposed hairs. The mature corollatube either abscises or may be removed by nectar-seeking non-flyingmarsupials, putative agents of pollination. Acrotriche serrulata, Epacridaceae, pollen presentation, pollination, geoflory, floral structure, nectar, stigma     

Microarray Analysis Reveals Similarities and Variations in Genetic Programs Controlling Pollination/Fertilization and Stress Responses in Rice (<Emphasis Type="Italic">Oryza sativa </Emphasis>L.)

Previously, we identified 253 cDNAs that are regulated by pollination/fertilization in rice by using a 10K cDNA microarray. In addition, many of them also appeared to be involved in drought and wounding responses. To investigate this relationship, we obtained their expression profiles after dehydration and wounding treatments in this study. Venn diagram analysis indicated that 53.8% (136/253) and 21% (57/253) of the pollination/fertilization-related genes are indeed regulated by dehydration and wounding, respectively, and nearly half of the genes expressed preferentially in unpollinated pistils (UP) are responsive to dehydration. These results indicated that an extensive gene set is shared among these responses, suggesting that the genetic programs regulating them are likely related. Among them, the genetic network of water stress control may be a key player in pollination and fertilization. Additionally, 39.5% (100/253) cDNAs that are related to pollination/fertilization appear not to be regulated by the stress treatments (dehydration and wounding), suggesting that the existence of additional genetic networks are involved in pollination/fertilization. Furthermore, comparative analysis of the expression profiles of the 253 cDNAs under 18 different conditions (various tissues, treatments and developmental status) revealed that the genetic networks regulating photosynthesis, starch metabolisms, GA- and defense-responses are involved in pollination and fertilization. Taken together, these results provided some clues to elucidate the molecular mechanisms of pollination and fertilization in rice. Supplementary material to this paper is available in electronic form at http://dx.doi.org/10.1007/s11103-005-3958-4     

Induction of 1-Aminocyclopropane-1-carboxylic Acid Synthase in Wounded Mesocarp Tissue of Winter Squash Fruit and the Effects of Ethylene

1-Aminocyclopropane-1-carboxylic acid (ACC) synthase activityincreased rapidly after wounding of mesocarp tissue of wintersquash fruit (Cucurbita maxima Duch.) and reached a peak at16 h after excision and then declined sharply. The rise in ACCsynthase activity was followed by increases in the endogenousACC content and the rate of ethylene production. The activityof ethylene forming enzyme (EFE) also increased rapidly in theexcised discs of mesocarp of winter squash fruit. ACC synthase activity was strongly inhibited by aminoethoxyvinylglycinewith a Ki value of 2.1 µM. Michaelis-Menten constant ofACC synthase for S-adenosylmethionine was 13.3 µM. Ethylene suppressed the induction of ACC synthase in the woundedmesocarp tissue. The suppression by ethylene increased withthe increasing concentrations of applied ethylene and the maximumeffect was obtained at about 100 µl 1^–1 ethylene,at which point the induction was suppressed by 54%. Ethylenedid not inhibit ACC synthase activity, nor did it suppress theinduction of EFE, but rather it slightly enhanced the latter. (Received August 24, 1984; Accepted October 29, 1984)     

The GUS reporter-aided analysis of the promoter activities of Arabidopsis ACC synthase genes AtACS4, AtACS5, and AtACS7 induced by hormones and stresses

Ethylene biosynthesis in higher plants is regulated developmentallyand environmentally. To investigate the regulation of ACC synthasegene expression, the promoters of Arabidopsis ACS genes, AtACS4,AtACS5, and AtACS7, were fused to a GUS reporter gene, and therecombinant transgenes were introduced into Arabidopsis to producethree groups of AtACS::GUS transgenic plants. Histochemic andfluorometric study of these transgenic plants revealed thatpromoters of AtACS4, AtACS, and AtACS7 are all active in dark-germinatedseedlings. AtACS5 has the highest promoter activity in leavesof 2-week-old light-grown seedlings among the three AtACS genesstudied. In the mature leaves, AtACS4 and AtACS7 genes are expressedin both veins and areoles, whereas AtACS5 is expressed at ahigher level in the areoles and epidermal cells surroundingtrichomes. The promoter activities of all these AtACS genesare found in the reproductive organs. AtACS5 and AtACS7 arehighly expressed in petals, sepals, carpels, stamens, caulineleaves, inflorescence stems, and siliques, while AtACS4 expressionis undetectable in the petals of open flowers. All three AtACSgenes are expressed in root tissue. In the 2-week-old light-grownArabidopsis, the AtACS4 promoter is responsive to the planthormones IAA, ethylene, and ABA, and to darkness and wounding;the AtACS5 promoter to IAA, ABA, salt, high temperature, andwounding; and the AtACS7 promoter to GA₃, ethylene, and ABA,and to darkness and salt. Low-temperature treatment abolishesthe darkness-induced AtACS7 gene expression, but not that ofAtACS4. Each AtACS gene has a unique expression profile duringgrowth and development. It appears that at any developmentalstage or any growth period of Arabidopsis, there is always amember of AtACS multigene family that is actively expressed. Key words: ACC synthase, Arabidopsis, ethylene, gene expression, GUS histochemical staining, reporter, stress treatments     

Early Events in the Penetration of the Embryo Sac in Torenia fournieri(Lind.)

At maturity, Torenia fournieri(Lind.) has an embryo sac whichprotrudes through the micropyle placing the synergids, egg celland part of the central cell within the ovary locule adjacentto the placenta. The present study utilized this unique attributein combination with confocal and light microscopy to characterizethe timing and associated structural changes during pollinationevents leading to double fertilization. The observation of spermnuclei in living gametophyte tissue is an important advancein the identification, in real time, of stages leading to fertilizationin angiosperms. A continuum of fertilization occurred between12 and 16 h after pollination (hap), with peak frequency ofegg and sperm fusion at 14 hap (43%). Movement of the spermcells through the degenerated synergid took several hours andfusion between sperm and their respective female nuclei occurredsimultaneously. Changes in embryo sac structure were also documented.Cell walls in the region between the synergids and egg cellwere poorly developed prior to pollen tube penetration. Thickenedcell walls were observed around the periphery of the synergidsand egg cell following pollination, and in the central cellwhere it lay within the body of the ovule. Starch was observedin the cells of the embryo sac, although the number and distributionof granules varied before and after pollination. These temporaland spatial observations of the embryo sac inTorenia fournieriprovide a basis for further research to determine control mechanismsoperating during specific double fertilization events in angiosperms.Copyright 2000 Annals of Botany Company Double fertilization, embryo sac, sperm nuclei, Hoechst, Torenia fournieri