Normal Pollen Germination Research Articles

FmRbohH Mediates ROS Generation and Enhances Pollen Tube Growth in Fraxinus mandshurica

Flowering plants require normal pollen germination and growth to be fertilized, but studies on the mechanism regulating pollen tube growth in Fraxinus mandshurica are limited. Here, we used transcriptomic data to study the oxidative phosphorylation pathway during pollen tube growth in Fraxinus mandshurica. Our study identified 8,734 differentially expressed genes during the stages S1 to S3 of pollen tube growth. Significant enrichment of the oxidative phosphorylation pathway, amino acid synthesis, protein processing in the ER, carbon metabolism, pyruvate metabolism, citrate cycle (TCA cycle), and glycolysis/gluconeogenesis were examined using the Kyoto Encyclopedia of Genes and Genomes, and 58 genes linked to ROS synthesis and scavenging during the S1–S3 stages were identified. Also, H2DCFDA staining confirmed ROS formation in the pollen and the pollen tubes, and treatment with copper (II) chloride (CuCl2) and diphenyleneiodonium (DPI) was shown to reduce ROS in the pollen tube. Reduction in ROS content caused decreased pollen germination and pollen tube length. Furthermore, FmRbohH (respiratory burst oxidase homolog H) expression was detected in the pollen and pollen tube, and an antisense oligodeoxynucleotide assay demonstrated reduced ROS and pollen tube growth in Fraxinus mandshurica. This study shed more light on the RbohH gene functions during pollen tube growth.

Roles of Pollen-Specific Boron Efflux Transporter, OsBOR4, in the Rice Fertilization Process

Arabidopsis thaliana BOR1 was the first boron (B) transporter identified in living systems. There are four AtBOR1-like genes, OsBOR1, 2, 3 and 4, present in the rice genome. We characterized the activity, expression and physiological function of OsBOR4. OsBOR4 is an active efflux transporter of B. Quantitative PCR analysis and OsBOR4 promoter-green fluorescent protein (GFP) fusion revealed that OsBOR4 was both highly and specifically expressed in pollen. We obtained five Tos17 insertion mutants of osbor4. The pollen grains were viable and development of floral organs was normal in the homozygous osbor4 mutants. We observed that in all Tos17 insertion lines tested, the frequency of osbor4 homozygous plants was lower than expected in the progeny of self-fertilized heterozygous plants. These results establish that OsBOR4 is essential for normal reproductive processes. Pollen from osbor4 homozygous plants elongated fewer tubes on wild-type stigmas, and tube elongation of mutant pollen was less efficient compared with the wild-type pollen, suggesting reduced competence of osbor4 mutant pollen. The reduced competence of mutant pollen was further supported by the crosses of independent Tos17-inserted alleles of OsBOR4. Our results suggest that OsBOR4, a boron efflux transporter, is required for normal pollen germination and/or tube elongation.

Post-Transcriptional Silencing of Flavonol Synthase mRNA in Tobacco Leads to Fruits with Arrested Seed Set

Flavonoids are synthesized by phenylpropanoid pathway. They are known to participate in large number of physiological and biochemical processes in plants. Parthenocarpy and male sterility has earlier been reported by silencing chalcone synthase (CHS) encoding gene. Silencing of CHS has blocked the synthesis of most of useful flavonoids including flavan-3-ols and flavonols. Also, these studies could not identify whether parthenocarpy/male sterility were due to lack of flavan-3-ols or flavonols or both. Flavonol synthase (FLS) is an important enzyme of flavonoid pathway that catalyzes the formation of flavonols. In this article, we propose a novel strategy towards the generation of seedless or less-seeded fruits by downregulation of flavonol biosynthesis in tobacco (Nicotiana tabacum cv Xanthi) through post-transcriptional gene silencing (PTGS) of FLS encoding mRNA. The FLS silenced lines were observed for 20-80% reduction in FLS encoding gene expression and 25–93% reduction in flavonol (quercetin) content. Interestingly, these FLS silenced tobacco lines also showed reduction in their anthocyanidins content. While the content of flavan-3-ols (catechin, epi-catechin and epi-gallocatechin) was found to be increased in FLS silenced lines. The delayed flowering in FLS silenced lines could be due to decrease in level of indole acetic acid (IAA) at apical region of their shoots. Furthermore, the pollen germination was hampered and pollens were unable to produce functional pollen tube in FLS silenced tobacco lines. Pods of FLS silenced lines contained significantly less number of seeds. The in vitro and in vivo studies where 1 µM quercetin was supplied to germination media, documented the restoration of normal pollen germination and pollen tube growth. This finding identified the role of flavonols particularly quercetin in pollen germination as well as in the regulation of plant fertility. Results also suggest a novel approach towards generation of seedless/less-seeded fruits via PTGS of FLS encoding gene in plants.

MALE GAMETOPHYTE DEFECTIVE 2, Encoding a Sialyltransferase‐like Protein, is Required for Normal Pollen Germination and Pollen Tube Growth in Arabidopsis

Sialyltransferases (SiaTs) exist widely in vertebrates and play important roles in a variety of biological processes. In plants, several genes have also been identified to encode the proteins that share homology with the vertebrate SiaTs. However, very little is known about their functions in plants. Here we report the identification and characterization of a novel Arabidopsis gene, MALE GAMETOPHYTE DEFECTIVE 2 (MGP2) that encodes a sialyltransferase-like protein. MGP2 was expressed in all tissues including pollen grains and pollen tubes. The MGP2 protein was targeted to Golgi apparatus. Knockout of MGP2 significantly inhibited the pollen germination and retarded pollen tube growth in vitro and in vivo, but did not affect female gametophytic functions. These results suggest that the sialyltransferase-like protein MGP2 is important for normal pollen germination and pollen tube growth, giving a novel insight into the biological roles of the sialyltransferase-like proteins in plants.

Reproductive biology of Boswellia serrata, the source of salai guggul, an important gum-resin

Detailed studies were carried out on the phenology, floral biology, pollination ecology and breeding system of Boswellia serrata Roxb. (Burseraceae) the source of ‘salai guggul’. The trees remain leafless during the entire period of flowering and fruiting. The inflorescence is a terminal raceme and produces up to 90 bisexual, actinomorphic flowers. On average a flower produces 10 044 ± 1259 starch-filled pollen grains. About 85% of the fresh pollen grains are viable; the pollen to ovule ratio is 3348 : 1. The stigma is of the wet papillate type. The style is hollow with three flattened stylar canals filled with a secretion product. The stylar canals are bordered by a layer of glandular canal cells. The inner tangential wall of the canal cells shows cellulose thickenings. The ovary is trilocular and bears three ovules, one in each locule. Flowers offer nectar and pollen as rewards to floral visitors. The giant Asian honey bee (Apis dorsata) and A. cerana var. indica(Indian honey bee) are the effective pollinators. The species is self-incompatible and the selfed pollen tubes are inhibited soon after their entry into the stigma. Self-pollen tubes develop a characteristic ‘isthmus’ as a result of enlargement of the tube soon after emergence through the narrow germ pore. Cross-pollinated flowers allowed normal pollen germination and pollen tube growth, and resulted in fruit- and seed-set. Under open pollination fruit-set was only about 10%. Although manual cross-pollinations increased fruit set, it was only up to about 20%. Low fruit set appears to be the result of inadequate cross-pollination and other constraints, presumably limitation of available nutrients. © 2005 The Linnean Society of London, Botanical Journal of the Linnean Society, 2005, 147, 73–82.

Biochemical complementation of chalcone synthase mutants defines a role for flavonols in functional pollen.

Chalcone synthase catalyzes the initial step of that branch of the phenylpropanoid pathway that leads to flavonoids. A lack of chalcone synthase activity has a pleiotropic effect in maize and petunia mutants: pollen fertility as well as flavonoid synthesis is disrupted. Both maize and petunia mutants are self-sterile due to a failure to produce a functional pollen tube. The finding that the mutant pollen is partially functional on wild-type stigmas led to the isolation and identification of kaempferol as a pollen germination-inducing constituent in wild-type petunia stigma extracts. We show that adding micromolar quantities of kaempferol to the germination medium or to the stigma at pollination is sufficient to restore normal pollen germination and tube growth in vitro and full seed set in vivo. Further we show that the rescue ability resides in particular structural features of a single class of compounds, the flavonol aglycones. This finding identifies another constituent of plant reproduction and suggests that addition or removal of the flavonol signal during pollen germination and tube growth provides a feasible way to control plant fertility.

Labile sex expression in andromonoecious Solanum hirtum: pattern of variation in floral structure

Comparison of hermaphrodite and staminate flowers of andromonoecious Solanum hirtum must consider two sources of morphological variation: variation due to differences in flower position and variation due to conditions that affect sex expression. In S. hirtum, positional variation occurs among hermaphrodite flowers of unpollinated plants; flowers vary quantitatively but not functionally with position on the inflorescence. On inflorescences of pollinated, fruit-bearing plants, flowers vary both quantitatively and functionally with position such that flowers borne distally are functionally staminate. Corolla, anther, style, and ovary length of staminate flowers are significantly smaller than those of hermaphrodite flowers and they have fewer, smaller ovules. Failure of fruit set in staminate flowers is due to a combination of factors. Stigmas and styles of staminate flowers are reduced and appear to lack the secretions necessary for normal pollen germination and growth. In the ovules, the integumentary tapetum is collapsed and appears senescent and most megagametophytes are abnormal. Lack of pollen tube growth and abnormal ovules could both contribute to gynoecial sterility in staminate flowers. Key words: andromonoecy, floral structure, reproductive anatomy, sex expression, Solanum.

The effect of ε-aminocaproic acid on self-incompatibility and incongruity inPhaseolus

SummaryThe effects of e-aminocaproic acid (EACA) on self-incompatibility was tested in a Phaseolus coccineus line which failed to form pods in controlled pollinations. Scanning electron microscope (SEM) and fluorescence miscroscopy confirmed problems in pollen-germination and pollen-tube growth but EACA-treated self- pollinations resulted in fairly normal pollen germination and pollen tube growth. In a few cases, pollen tubes were seen entering the embryo sac and occasionally pods were formed. Incongruity barriers also exist in crosses of P. coccineus × P. vulgaris, P. acutifolius × P. coccineus and their reciprocals. Effects of EACA on flower abscission, pod development and pod abscission were studied. Increased pod formation was observed in EACA treated materials, except in P. acutifolius × P. coccineus and P. coccineus × P. vulgaris cv. Jacobs Cattle. EACA seems to act at the stigmatic and stylar levels, thereby enhancing pollen germination and pollen tube growth and delaying flower abscission. The net...

Intersubgeneric Crossability in the Genus Glycine Willd.

AbstractIn intersubgeneric crosses between wild perennial Glycine species of submenus Glycine and soybeans, Glycine max (L.) Merr., of subgenus Soja, all the putative hybrid pods aborted from 9 to 30 days after pollination (DAP). Spraying of 70 ppm gibberellic acid on hybridized gynoecia facilitated pod retention. Fluorescence microscopy revealed normal pollen germination and pollen tube growth in all the intersubgeneric crosses except in the G. cyrtoloba × G. max cross.

POLLEN LONGEVITY AND STIGMA PRE‐EMPTION IN CLARKIA

The ability of pollen to effect seed set decreases with age when stored at room temperature or on the plant, but not when stored at 5 C. Seed set declines rapidly with pollen stored 10 days or more at room temperature. Seed set declines rapidly with pollen stored 4 days or more on the plant. The degree of pollen germination on the stigma and the number of pollen tubes in the style correlate positively with seed set. Pollen germination on the stigma and pollen tube growth in the style are strongly correlated with seed set for pollen stored at room temperature, but not, or only weakly, correlated for pollen stored at 5 C. In the latter case, seed sets are lower than expected, indicating a possible viability reduction associated with a post‐pollination function. Inviable pollen grains temporarily interfere with normal pollen germination on the stigma. Essentially no seeds are produced when in viable pollen, immediately followed by viable pollen, is placed on a fresh stigma. However 27 to 65 seeds are produced when in viable pollen, followed in 1 to 6 days by viable pollen, is placed on fresh stigmas. New stigma surface is exposed at the center as expanding stigma surfaces carry the interfering inviable pollen toward the edge of the stigma.

Membrane-associated calcium during pollen grain germination: a microfluorometric analysis

Pear (Pyrus communis L.) pollen was germinated and grown in hanging drop cultures containing phenothiazine drugs, trifluoperazine and chlorpromazine, potent inhibitors of the Ca2+-calmodulin complex. Responses for the two drugs were similar: at 1.0–2 10.0 ΜM pollen germination and tube growth were inhibited. Inhibition of tube growth was not uniform; at 10.0 ΜM growth of about half of those tubes which had germinated was inhibited while the remaining half of the population grew normally. This bimodal distribution of tube growth was noted, but to a lesser extent, at lower concentrations of the drugs as well. Microfluorometric analysis of membrane calcium using chlorotetracycline, a fluorescent chelate probe for membrane calcium, revealed that upon pollen grain hydration there was a sharp decrease in the concentration of calcium associated with membranes and that membrane calcium became localized primarily at the periphery of the newly hydrated pollen grain. Prior to germination there was a reloading of calcium onto membranes in the region of the germination aperture through which the pollen tube would emerge. The release of Ca2+ from membrane sites upon hydration was partially inhibited by treatment with 10 ΜM trifluoperazine. Distribution of membrane calcium in populations of the inhibitor-treated pollen grains was not bimodal however; approximately half of the population had CTC-fluorescence emissions exceeding the maximum value found in the control population. These results suggest that there is a maximum concentration of membrane-associated calcium consistent with normal pollen germination and tube growth and that phenothiazines interfere with the unloading of Ca2+ from membrane sites.

THE ROLES OF POLYEMBRYONY AND EMBRYO VIABILITY IN THE GENETIC SYSTEM OF CONIFERS.

The mating and regeneration habits of many important coniferous species seem conducive to self pollination, exchange of pollen among relatives in small family groups, and relatively high levels of inbreeding. Most species show large inbreeding depression in growth, and the frequency of self seedlings in wind-pollination progeny is generally low. In this paper, I will show that low embryo survival plus the presence of more than one embryo in a single ovule (polyembryony) allows considerable post-fertilization embryo abortion and selection without comparable wastage of ovules. In combination they form an important mechanism for maintaining heterozygosity as well as local adaptation. Self incompatibility, in the sense of blockage to pollen tube penetration and selective fertilization (Squillace and Bingham, 1958), could perform a similar function. It also would contribute to maintaining heterozygosity in the presence of high frequencies of self pollination. Experimental evidence for self incompatibility in conifers is lacking (Hagman and Mikkola, 1963; Hagman, 1972) but it can not be ruled out at this point. If selective fertilization occurs it could further augment the effect of polyembryony. Low viability of inbred embryos, a feature of many of the widespread coniferous species, is attributed to recessive lethal and deleterious alleles which become homozygous after inbreeding (Orr-Ewing, 1957; Sarvas, 1962; Hagman and Mikkola, 1963; Mergen et al., 1965). Many of these alleles apparently are present at low frequencies in the breeding populations (Koski, 1971). Cytological examinations have shown embryo abortion to occur early in embryogeny after apparently normal pollen germination and fertilization (OrrEwing, 1957; Hagman and Mikkola, 1963; Mergen et al., 1965; Sarvas, 1968). Polyembryony, a rare derivative phenomenon in angiosperms but unusually common in gymnosperms (Chamberlain, 1966, p. 348), is of two types. One is categorized as simple, archegonial, polyzygotic, or non-cleavage, and the other as monozygotic or cleavage. Archegonial polyembryony, characterized by independent fertilization of more than one archegonium within an ovule, gives rise to heterogenic embryos within the ovule. Within the family Pinaceae, it occurs in the genera Larix, Picea, and Pseudotsuga (Dogra, 1967, p. 17). Monozygotic polyembryony is characterized by cleavage of the embryo and gives rise to isogenic embryos. Both monoand polyzygotic polyembryony occur in the genera Pinus and Tsuga (Dogra, 1967, p. 17, 83). The situation is not clear in Abies, which is classified as having non-cleavage polyembryony by Dogra (1967, p. 16); but in Abies amabilis at least there appear to be more embryos than could arise from noncleavage polyembryony alone (Owens and Molder, 1977), which indicates a pattern like Pinus and Tsuga. Polyembryony eliminates the effects of aborted embryo development when the abortion occurs in an ovule that contains an additional functional embyro to replace the degenerating one. Also, because one embryo usually ends up dominant in the embryo cavity, polyembryony provides opportunity for selection between embryos of unequal vigor during early embryo development. Embryological development can be climatically as well as genetically disturbed. Monozygotic polyembryony has the potential for reducing the effects of climatic disturbance; archegonial polyembryony has the potential for reducing

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

Stigma-surface esterase activity and stigma receptivity through a sequence of developmental stages of the pistil have been studied in four taxa characterized by having wet stigmas-Petunia hybrida, Nicotiana tabacum, Crinum defixum and Amaryllis vittata. The style is solid in the first two and hollow in the latter two taxa. In all the taxa, stigma-surface esterase could be detected in a thin surface layer (pellicle) from a very early stage of pistil development, irrespective of the presence or absence of the exudate. However, the taxa showed variation in stigma receptivity. In Petunia and Nicotiana, stigmas from pistils of 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 normal pollen germination and tube growth. It is inferred that in taxa characterized by a wet stigma and solid style, the factors required for pollen germination are present from an early stage of pistil development and the exudate per se is not involved in pollen germination. In taxa characterized by a wet stigma and hollow style, however, the pellicle does not carry the factors required for pollen germination and tube growth; they appear to be present in the exudate.