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PubMed1985

Applications of fluorochromes to pollen biology. I. Mithramycin and 4',6-diamidino-2-phenylindole (DAPI) as vital stains and for quantitation of nuclear DNA.

The two DNA-specific fluorochromes DAPI and mithramycin have been found to be extremely useful dyes in studies of pollen development and growth. Both fluorochromes stain nuclei brilliantly either in fixed or in living tricellular and bicellular angiosperm pollen, thereby permitting rapid scanning for pollen abnormalities and easy observation of nuclear details. These water soluble dyes can be incorporated into the germination medium for studies of pollen germination in vitro, facilitating observation of the movement of generative, sperm and tube nuclei during pollen growth. In fixed pollen, the fluorochromes bind quantitatively with DNA and thus may be used to quantitate ploidy changes and to study cell cycles during pollen development, germination and fertilization.

DNA↗
PubMed1981

Studies on heteromorphic self-incompatibility systems: the cytochemistry and ultrastructure of the tapetum of Primula obconica.

This paper describes the cytochemistry and ultrastructure of the developing tapetum in Primula obconica, a plant with a heteromorphic, sporophytic self-incompatibility system. The tapetum is of the secretory type and cytochemical tests have shown that when it breaks down proteinaceous (esterase) and lipidic components are deposited on the developing pollen grains. Acid phosphatase, a marker of gametophytic enzyme activity, is confined to the cytoplasm and intine of the developing pollen. Ultrastructural studies show that prior to its dissolution the tapetum undergoes a number of changes. In the early stages of development the tapetum is rich in ribosomes and rough endoplasmic reticulum, but following the breakdown of the tapetal cell wall the main components of the cytoplasm are densely staining spherical bodies surrounded by ribosomes and orbicular bodies, which appear to be confined to the cell periphery. As the cells break down, rod-like fibrils can be seen amongst the degenerate organelles and within the bacular cavities of the pollen. On dehiscence the pollen has a lipidic coating in addition to the fibrillar material in the pollen wall and the remnants of the tapetum can be seen adhering to the fibrous layer of the anther wall. Thus the mature, binucleate pollen of P. obconica is demonstrated to carry wall materials of sporophytic origin.

Acid Phosphatase↗
PubMed2004

Programmed cell death induces male sterility in Actinidia deliciosa female flowers.

The importance of programmed cell death (PCD) during the life cycle of plants is well established, although the underlying molecular mechanisms are still poorly defined. An emerging system for the study of PCD during development in plants is that of sex organ abortion. In this work we investigate the degeneration of microspores in the anthers of Actinidia deliciosa female flowers. The kiwifruit, A. deliciosa, is a dioecious species native to China. Pollen development in female flowers is equivalent to pollen development in the male flowers, until the microspores are released from the tetrads. At this time the first differences appear, and include the condensation and shrinkage of the cytoplasm, blebbing of the plasma membrane and of the nuclear envelope, and condensation of chromatin. However, at the time these events are occurring, all other cellular organelles, including mitochondria, have their structures well preserved. Fragmentation of DNA was detected in situ by the TUNEL procedure, which involves the end labeling of the DNA fragments by terminal deoxynucleotidyl transferase with UTP conjugated to a detectable marker. This assay confirmed the morphological characterization of PCD in this system.

Actinidia↗
PubMed1996

A cytoplasmic male sterility-associated mitochondrial protein causes pollen disruption in transgenic tobacco.

In higher plants, dominant mitochondrial mutations are associated with pollen sterility. This phenomenon is known as cytoplasmic male sterility (CMS). It is thought that the disruption in pollen development is a consequence of mitochondrial dysfunction. To provide definitive evidence that expression of an abnormal mitochondrial gene can interrupt pollen development, a CMS-associated mitochondrial DNA sequence from common bean, orf239, was introduced into the tobacco nuclear genome. Several transformants containing the orf239 gene constructs, with or without a mitochondrial targeting sequence, exhibited a semi sterile or male-sterile phenotype. Expression of the gene fusions in transformed anthers was confirmed using RNA gel blotting, ELISA, and light and electron microscopic immunocytochemistry. Immunocytological analysis showed that the ORF239 protein could associate with the cell wall of aberrant developing microspores. This pattern of extracellular localization was earlier observed in the CMS common bean line containing orf239 in the mitochondrial genome. Results presented here demonstrate that ORF239 causes pollen disruption in transgenic tobacco plants and may do so without targeting of the protein to the mitochondrion.

Chimera↗
PubMed2005

Callose (beta-1,3 glucan) is essential for Arabidopsis pollen wall patterning, but not tube growth.

BACKGROUND: Callose (beta-1,3 glucan) separates developing pollen grains, preventing their underlying walls (exine) from fusing. The pollen tubes that transport sperm to female gametes also contain callose, both in their walls as well as in the plugs that segment growing tubes. Mutations in CalS5, one of several Arabidopsis beta-1,3 glucan synthases, were previously shown to disrupt callose formation around developing microspores, causing aberrations in exine patterning, degeneration of developing microspores, and pollen sterility. RESULTS: Here, we describe three additional cals5 alleles that similarly alter exine patterns, but instead produce fertile pollen. Moreover, one of these alleles (cals5-3) resulted in the formation of pollen tubes that lacked callose walls and plugs. In self-pollinated plants, these tubes led to successful fertilization, but they were at a slight disadvantage when competing with wild type. CONCLUSION: Contrary to a previous report, these results demonstrate that a structured exine layer is not required for pollen development, viability or fertility. In addition, despite the presence of callose-enriched walls and callose plugs in pollen tubes, the results presented here indicate that callose is not required for pollen tube functions.

Alleles↗
PubMed2003

Identification of higher plant GlsA, a putative morphogenesis factor of gametic cells.

GlsA has been identified in an asexual-reproductive-cell (gonidia)-deficient mutant of Volvox as a chaperone-like protein essential for gonidia production. In this study, we isolated an angiosperm glsA (LlglsA) gene expressed during Lilium longiflorum pollen development. Immunoblot analyses showed that the strong LlGlsA expression occurred in the generative cell and its pattern during pollen development corresponded to that of alpha-tubulin. Morphological analyses succeeded in visualizing the dispersion of the strong LlGlsA signal in developing generative cells. In addition, multiple-immunofluorescence staining of LlGlsA and alpha-tubulin revealed that some of the dot-like LlGlsA signals were co-localized with microtubule filaments. From those results, we suggest that angiosperm GlsA functions as a chaperone modifying various structures during male gametic cell formation.

Algal Proteins↗
PubMed1994

Characterization of a pollen-expressed receptor-like kinase gene of Petunia inflata and the activity of its encoded kinase.

From a pollen tube cDNA library of Petunia inflata, we isolated clones encoding a protein with structural features and biochemical properties characteristic of receptor-like kinases. It was designated PRK1 for pollen receptor-like kinase 1. The cytoplasmic domain of PRK1 is highly similar to the kinase domains of other plant receptor-like kinases and contains nearly all of the conserved amino acids for serine/threonine kinases. The extracellular domain of PRK1 contains leucine-rich repeats as found in some other plant receptor-like kinases, but overall its sequence in this region does not share significant similarity. Characterization of a gene encoding PRK1 revealed the presence of two introns. During pollen development, PRK1 mRNA was first detected in anthers containing mostly binucleate microspores; it reached the highest level of mature pollen and remained at a high level in in vitro-germinated pollen tubes. The recombinant cytoplasmic domain of PRK1 autophosphorylated on serine and tyrosine, suggesting that PRK1 may be a dual-specificity kinase. Monospecific immune serum to the recombinant extracellular domain of PRK1 detected a 69-kD protein in microsomal membranes of pollen and pollen tubes. The characteristics of PRK1 suggest that it may play a role in signal transduction events during pollen development and/or pollination.

Amino Acid Sequence↗
PubMed2004

Isolation and characterization of a rice cysteine protease gene, OsCP1, using T-DNA gene-trap system.

The T-DNA gene-trap system has been efficiently used to elucidate gene functions in plants. We report here a functional analysis of a cysteine protease gene, OsCP1, isolated from a pool of T-DNA insertional rice. GUS assay with the T-DNA tagged line indicated that the OsCP1 promoter was highly active in the rice anther. Sequence analysis revealed that the deduced amino acid sequence of OsCP1 was homologous to those of papain family cysteine proteases containing the highly conserved interspersed amino acid motif, ERFNIN. This result suggested that the gene encodes a cysteine protease in rice. We also identified a suppressed mutant from T2 progeny of the T-DNA tagged line. The mutant showed a significant defect in pollen development. Taken together, the results demonstrated that OsCP1 is a cysteine protease gene that might play an important role in pollen development.

Amino Acid Sequence↗
PubMed2002

The 5'-untranslated region of the ntp303 gene strongly enhances translation during pollen tube growth, but not during pollen maturation.

Transcripts of the ntp303 gene accumulate abundantly throughout pollen development, whereas the protein only accumulates to detectable levels after pollen germination. In an attempt to explain the divergence in the accumulation profiles of the mRNA and the protein, we investigated the role of the untranslated regions (UTRs) in enhancing ntp303 translation during the transition from developing to germinating pollen. Luciferase reporter gene fusion constructs containing the ntp303 5'-UTR gave rise to luciferase activity that was up to 60-fold higher during pollen tube growth than that of constructs containing different 5'-UTRs. No apparent differences in the luciferase activity of these constructs were observed during pollen development. The ntp303 5'-UTR-mediated increase in luciferase activity was not significantly influenced by coding region or 3'-UTR sequences. Furthermore, enhanced luciferase activity directed by the ntp303 5'-UTR occurred predominantly at the post-transcriptional level. A series of 5'-UTR deletion constructs was created to identify putative regulatory sequences required for the high level of translation during pollen tube growth. Two predicted stem loop structures (H-I and H-II) caused a complete inhibition of the enhanced translation after their total or partial deletion. A (GAA)(8) repeat within the H-I stem loop structure was demonstrated to be important for the modulation of translation efficiency. The H-II stem loop structure was found to be essential for the determination of mRNA stability.

3' Untranslated Regions↗
PubMed2003

Arabidopsis AtGPAT1, a member of the membrane-bound glycerol-3-phosphate acyltransferase gene family, is essential for tapetum differentiation and male fertility.

Membrane-bound glycerol-3-phosphate acyltransferase (GPAT; EC 2.3.1.15) mediates the initial step of glycerolipid biosynthesis in the extraplastidic compartments of plant cells. Here, we report the molecular characterization of a novel GPAT gene family from Arabidopsis, designated AtGPAT. The corresponding polypeptides possess transmembrane domains and GPAT activity when expressed heterologously in a yeast lipid mutant. The functional significance of one isoform, AtGPAT1, is the focus of the present study. Disruption of the AtGPAT1 gene causes a massive pollen development arrest, and subsequent introduction of the gene into the mutant plant rescues the phenotype, illustrating a pivotal role for AtGPAT1 in pollen development. Microscopic examinations revealed that the gene lesion results in a perturbed degeneration of the tapetum, which is associated with altered endoplasmic reticulum profiles and reduced secretion. In addition to the sporophytic effect, AtGPAT1 also exerts a gametophytic effect on pollen performance, as the competitive ability of a pollen grain to pollinate is dependent on the presence of an AtGPAT1 gene. Deficiency in AtGPAT1 correlates with several fatty acid composition changes in flower tissues and seeds. Unexpectedly, however, a loss of AtGPAT1 causes no significant change in seed oil content.

Amino Acid Sequence↗
PubMed2006

Study of male sterility in Taiwania cryptomerioides Hayata (Taxodiaceae).

A study of male sterility over a period of three consecutive years on a conifer species endemic to Taiwan, Taiwania cryptomerioides Hayata (Taxodiaceae), was done for this article. With the aids of fluorescence and electron microscopic observations, the ontogenic processes in the fertile and sterile microsporangia are compared, using samples collected from Chitou Experimental Forest and Yeou-Shoei-Keng Clonal Orchard of the National Taiwan University, Nantou, Taiwan. The development of male strobili occurred from August to the end of March. Microsporogenesis starts with the formation of the archesporium and ends with the maturation of 2-celled pollen grains within the dehiscing microsporangium. Before meiosis, there was no significant difference in ultrastructure between the fertile and sterile microsporangia. Asynchronous pollen development with various tetrad forms may occur in the same microsporangium of either fertile or sterile strobili. However, a callose wall was observable in the fertile dyad and tetrad, but not in the sterile one. After dissolution of the callose wall, the fertile microspores were released into the locule, while some sterile microspores still retained as tetrads or dyads with intertwining of exine walls in the proximal faces. As a result, there was no well developed lamellated endexine and no granulate ectexine or intine in the sterile microspores. Eventually, the intracellular structures in sterile microspores were dramatically collapsed before anthesis. The present study shows that the abortion in pollen development is possibly attributed to the absence of the callose wall. The importance of this structure to the male sterility of T. cryptomerioides is discussed.

Cupressaceae↗
PubMed1990

Characterization of a gene family abundantly expressed in Oenothera organensis pollen that shows sequence similarity to polygalacturonase.

We have isolated and characterized cDNA clones of a gene family (P2) expressed in Oenothera organensis pollen. This family contains approximately six to eight family members and is expressed at high levels only in pollen. The predicted protein sequence from a near full-length cDNA clone shows that the protein products of these genes are at least 38,000 daltons. We identified the protein encoded by one of the cDNAs in this family by using antibodies to beta-galactosidase/pollen cDNA fusion proteins. Immunoblot analysis using these antibodies identifies a family of proteins of approximately 40 kilodaltons that is present in mature pollen, indicating that these mRNAs are not stored solely for translation after pollen germination. These proteins accumulate late in pollen development and are not detectable in other parts of the plant. Although not present in unpollinated or self-pollinated styles, the 40-kilodalton to 45-kilodalton antigens are detectable in extracts from cross-pollinated styles, suggesting that the proteins are present in pollen tubes growing through the style during pollination. The proteins are also present in pollen tubes growing in vitro. Both nucleotide and amino acid sequences are similar to the published sequences for cDNAs encoding the enzyme polygalacturonase, which suggests that the P2 gene family may function in depolymerizing pectin during pollen development, germination, and tube growth. Cross-hybridizing RNAs and immunoreactive proteins were detected in pollen from a wide variety of plant species, which indicates that the P2 family of polygalacturonase-like genes are conserved and may be expressed in the pollen from many angiosperms.

Amino Acid Sequence↗
PubMed2006

Characterization and expression of Arabidopsis UDP-sugar pyrophosphorylase.

At5g52560, a homolog of pea (Pisum sativum) UDP-sugar pyrophosphorylase (PsUSP) was functionally annotated by expression in Escherichia coli and subsequent characterization of substrate specificity and kinetic properties. Arabidopsis contains a single USP gene (AtUSP) and evaluation of gene databases suggests that USP is unique to plants. The 69 kDa AtUSP gene product exhibited high activity with Glc-1-P, GlcA-1-P and Gal-1-P, but low activity with GlcNAc-1-P, Fuc-1-P, Man-1-P, inositol-1-P or Glc-6-P. AtUSP was activated by magnesium and preferred UTP as co-substrate. Apparent K(m) values for GlcA-1-P, Glc-1-P and UTP were 0.13 mM, 0.42 mM and 0.14 mM, respectively. In the reverse direction (pyrophosphorolysis), the apparent K(m) values for UDP-GlcA, UDP-Glc and pyrophosphate were 0.56 mM, 0.72 mM and 0.15 mM, respectively. USP enzyme activity (UDP-GlcA --> GlcA-1-P) was detected in Arabidopsis tissues with highest activity found in the inflorescence. As determined by semi-quantitative RT-PCR, AtUSP transcript is widely expressed with high levels detected in the inflorescence. To evaluate tissue-specific expression of AtUSP, histochemical GUS staining of plants transformed with AtUSPprom:GUS constructs was performed. In 7-day-old seedlings, GUS staining was detected in cotyledons, trichomes and vascular tissues of the primary root. In the inflorescence of older plants, high levels of GUS staining were detected in cauline leaves, the epidermis of the stem and in pollen. In silico analysis of AtUSP expression in developing pollen indicates that transcript levels increase as development proceeds from the uninucleate to the tricellular stage. The results suggest that AtUSP plays an important role in pollen development in Arabidopsis.

Arabidopsis↗
PubMed2004

Analysis of transposon insertion mutants highlights the diversity of mechanisms underlying male progamic development in Arabidopsis.

To identify genes with essential roles in male gametophytic development, including postpollination (progamic) events, we have undertaken a genetic screen based on segregation ratio distortion of a transposon-borne kanamycin-resistance marker. In a population of 3359 Arabidopsis Ds transposon insertion lines, we identified 20 mutants with stably reduced segregation ratios arising from reduced gametophytic transmission. All 20 mutants showed strict cosegregation of Ds and the reduced gametophytic transmission phenotype. Among these, 10 mutants affected both male and female transmission and 10 mutants showed male-specific transmission defects. Four male and female (ungud) mutants and 1 male-specific mutant showed cellular defects in microspores and/or in developing pollen. The 6 remaining ungud mutants and 9 male-specific (seth) mutants affected pollen functions during progamic development. In vitro and in vivo analyses are reported for 5 seth mutants. seth6 completely blocked pollen germination, while seth7 strongly reduced pollen germination efficiency and tube growth. In contrast, seth8, seth9, or seth10 pollen showed reduced competitive ability that was linked to slower rates of pollen tube growth. Gene sequences disrupted in seth insertions suggest essential functions for putative SETH proteins in diverse processes including protein anchoring, cell wall biosynthesis, signaling, and metabolism.

Arabidopsis↗
PubMed2005

Tall tales from sly dwarves: novel functions of gibberellins in plant development.

Gibberellins (GAs) are endogenous hormones controlling numerous aspects of plant growth and development. Our present understanding of GA physiology is based largely on genetic analysis in model plants such as Arabidopsis. In spite of the success of this approach, the discovery of additional physiological roles for GAs in seed development, pollen tube growth and meristem development indicates that the existing collection of GA-related mutants (identified partially or entirely on the basis of vegetative phenotypes) has failed to uncover all aspects of plant development that are controlled by GAs. The continued use of ever improving forward and reverse genetic techniques is expected to lead to the discovery of further novel roles for GAs in plant development.

Arabidopsis↗
PubMed1994

Cloning and characterization of Tag 1, a tobacco anther beta-1,3-glucanase expressed during tetrad dissolution.

A critical stage in pollen development is the dissolution of the four products of meiosis, the tetrads, into free microspores. The tetrads are surrounded by a thick callose wall composed of beta-1,3-glucan. At the completion of meiosis, the tetrads are released into the anther locule after hydrolysis of the callose by a beta-1,3-glucanase. Using the polymerase chain reaction, we have amplified and subsequently cloned a cDNA corresponding to a beta-1,3-glucanase, tobacco (Nicotiana tabacum cv. Samsun) anther glucanase (Tag 1), which is expressed exclusively in anthers from meiosis to the free microspore stage of pollen development. The identity of the clone was determined by DNA and deduced protein sequence similarity to other known beta-1,3-glucanases. Several regions strictly conserved among four classes of glucanases are also conserved in the Tag 1 protein. Tag 1 represents a novel class of beta-1,3-glucanase based on phylogenetic analysis and RNA expression pattern. Tag 1 RNA was detected in situ only in the tapetum, with maximal expression just prior to tetrad dissolution. Due to its expression pattern and sequence similarity to other beta-1,3-glucanases, we believe Tag 1 may be involved in tetrad dissolution.

Amino Acid Sequence↗