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PubMed1992

A Brassica napus gene family which shows sequence similarity to ascorbate oxidase is expressed in developing pollen. Molecular characterization and analysis of promoter activity in transgenic tobacco plants.

The genomic clone named Bp10 contains a member of a small pollen-specific gene family of B. napus. The expression of the Bp10 gene family is maximal in early binucleate microspores and declines considerably in mature trinucleate pollen. Homologues of the Bp10 genes are expressed in the pollen of other plant species. The pollen-specific expression of the gene contained in the genomic clone was confirmed in tobacco plants transformed with a chimeric Bp10 promoter/GUS construct. A promoter fragment of 396 bp is sufficient to direct a strong and correct spatial and temporal expression in transgenic plants. The Bp10 gene family codes for proteins of 62 kDa showing approximately 30% sequence identify to cucumber and pumpkin ascorbate oxidases (AAOs). However, the AAO active centres are not conserved in the Bp10 products, suggesting an evolutionary relationship but a different enzymatic activity for these proteins. Expression of a recombinant Bp10 protein in E. coli inhibits bacterial growth on minimal medium, suggesting the production of an enzymatically active polypeptide in bacteria. No AAO activity could be correlated with the expression of the recombinant protein. Moreover, substances affecting AAO activity do not appear to influence the inhibitory activity of the protein produced in bacteria. However, as indicated by the rescue of bacterial growth in the presence of sodium bicarbonate or gaseous CO2, the Bp10 protein activity could be modulated by CO2 levels.

Amino Acid Sequence↗
PubMed1999

Identification of a pollen-specific sucrose transporter-like protein NtSUT3 from tobacco.

Pollen cells are symplasmically isolated during maturation and germination. Pollen therefore needs to take up nutrients via membrane carriers. Physiological measurements on pollen indicate sucrose transport in the pollen tube. A cDNA encoding a pollen-specific sucrose transporter-like protein NtSUT3 was isolated from a tobacco pollen cDNA library. NtSUT3 expression is detected only in pollen and is restricted to late pollen development, pollen germination and pollen tube growth. Altogether these data indicate that pollen is supplied not only with glucose, but also with sucrose through a specific sucrose transporter. The respective contribution of each transport pathway may change during pollen tube growth.

Amino Acid Sequence↗
PubMed1997

Cloning and characterization of a pollen-specific cDNA encoding a glutamic-acid-rich protein (GARP) from potato Solanum berthaultii.

A pollen-specific cDNA was isolated from a cDNA library of in vitro germinated pollen of the diploid potato species Solanum berthaultii. The cDNA clone, designated SB401, hybridizes to a messenger RNA of 1.2 kb length in mature and germinated pollen. SB401 messenger RNA is absent from other parts of the plant, including other flower tissues. SB401 cDNA, which possesses a long stretch of AT-rich 5'-untranslated leader sequence, encodes a glutamic acid-rich protein (GARP) which is hydrophilic throughout and contains six imperfect repeated motifs of the sequence V-V-E-K-K-N/E-E with the di-basic amino acid residue pair (K-K) as the core within the repeats. These repeats are spaced at irregular intervals and predicted to form an alpha-helical structure. The SB401 protein was over-expressed in Escherichia coli and the purified protein was used for raising antiserum. Both E. coli-expressed and the endogenous SB401 proteins in pollen and pollen tubes appear much larger on SDS-polyacrylamide gels than their calculated molecular masses. Immunoblotting revealed the protein to be most abundant in germinated pollen. The structural features of SB401 protein and a possible role for the protein in pollen development, pollen germination, and pollen tube growth are discussed.

Amino Acid Sequence↗
PubMed1997

Regulation of flavonol biosynthesis during anther and pistil development, and during pollen tube growth in Solanum tuberosum.

The regulation of flavonol biosynthesis was studied in anthers and pistils of Solanum tuberosum. Flavonols are essential for functional pollen tube growth in a number of species. Flavonol accumulation in whole anthers started at the unicellular stage of pollen development and continued until pollen maturity. A cDNA clone encoding flavonol synthase (FLS) was isolated. Fls gene expression was detected in pistils, anthers, petals and ovaries, the organs in which flavonols are accumulating. Fls transcripts were present in unicellular and bicellular pollen, but not in mature pollen. The expression patterns of three genes encoding enzymes in the flavonoid biosynthetic pathway, chalcone synthase (chs), flavanone-3-hydroxylase and fls were analysed in developing anthers and pistils. Only chs transcripts accumulated concomitantly with the flavonols in anthers. In pistils of potato, pollen tube growth induced an increase in fls gene expression that, unlike the situation in pollinated pistils of petunia, did not result in an increased flavonol content. Flavonol biosynthesis in anthers is probably initiated by the expression of the chs gene, and flavonol accumulation in pistils upon pollen tube growth is not an universal phenomenon.

Acyltransferases↗
PubMed1999

Localization of the Ca(2+)-binding protein, Bra r 1, in anthers and pollen tubes.

Calcium plays an essential role during pollen development and pollen tube growth, and several Ca(2+)-binding proteins are expressed in anthers. We have previously reported that Brassica pollen allergens encoded by Bra r 1 and Bra r 2 show sequence similarities to Ca(2+)-binding proteins [Toriyama et al. (1995) Plant Mol. Biol. 29: 1157]. Herein, we report that both genes are expressed in the diploid tapetum and haploid microspores, as detected by in situ RNA hybridization. Immunoblot analysis revealed that Bra r 1 and Bra r 2 were accumulated in anthers during pollen development. When pollen grains were suspended in an aqueous solution, both proteins were mainly detected in the pollen extracellular fraction, indicating that Bra r 1 and Bra r 2 are released from the pollen upon hydration. Localization of Bra r 1 was further investigated in sections of anthers and pollen tubes. Bra r 1 was detected in the tapetum, microspores and pollen grains. In longitudinal sections of cross-pollinated pistils. Bra r 1 was detected throughout pollen tubes elongating in transmitting-tissue. These findings suggest that Bra r 1 may be involved in pollen-pistil interaction and pollen tube growth.

Allergens↗
PubMed1994

Hsp70 heat shock protein cognate is expressed and stored in developing tomato pollen.

Pollen of angiosperms lacks the ability to respond to heat stress by synthesizing heat shock proteins (hsps). In tomato developing microspores were found to have 70 kDa heat shock proteins (hsp70s) present throughout development, even in the absence of heat stress. Heat shock protein family members expressed in the absence of heat stress are called cognate (hsc70) genes. Antisense RNA and antibody probes were used for in situ hybridizations which detected hsc70 expression in developing pollen of immature buds. Hsc70 mRNA transcripts and proteins were detected in nonstressed sporogenous tissues, microspores and in pre-tapetal layers during early pollen development. While immunoblot analysis detected hsc70 proteins stored in mature pollen, heat stress could not induce the synthesis of new hsp70 protein as measured by 35S-methionine labeling followed by immunoprecipitation.

HSP70 Heat-Shock Proteins↗
PubMed2003

A calmodulin-binding protein from Arabidopsis has an essential role in pollen germination.

Calmodulin (CaM), a ubiquitous multifunctional calcium sensor in all eukaryotes, mediates calcium action by regulating the activity/function of many unrelated proteins. Although calcium and CaM are known to play a crucial role in pollen germination and pollen tube growth, the proteins that mediate their action have not been identified. We isolated three closely related CaM-binding proteins (NPG1, NPGR1, and NPGR2) from Arabidopsis. NPG1 (No Pollen Germination1) is expressed only in pollen, whereas the NPG-related proteins (NPGR1 and NPGR2) are expressed in pollen and other tissues. The bacterially expressed NPG1 bound three isoforms of Arabidopsis CaM in a calcium-dependent manner. To analyze the function of NPG1, we performed a reverse genetics screen and isolated a mutant in which NPG1 is disrupted by a T-DNA insertion. Segregation and molecular analyses of the NPG1 knockout mutant and a cross with a male sterile mutant indicate that the mutated NPG1 is not transmitted through the male gametophyte. Expression of NPG1 in the knockout mutant complemented the mutant phenotype. Analysis of pollen development in the knockout mutant by light microscopy showed normal pollen development. Pollen from NPG1 mutant in the quartet background has confirmed that NPG1 is dispensable for pollen development. However, germination studies with pollen from the mutant in the quartet background indicate that pollen carrying a mutant allele does not germinate. Our genetic, histological, and pollen germination studies with the knockout mutant line indicate that NPG1 is not necessary for microsporogenesis and gametogenesis but is essential for pollen germination.

Alleles↗
PubMed1997

TETRASPORE is required for male meiotic cytokinesis in Arabidopsis thaliana.

In flowering plants, male meiosis occurs in the microsporocyte to produce four microspores, each of which develops into a pollen grain. Here we describe four mutant alleles of TETRASPORE (TES), a gene essential for microsporocyte cytokinesis in Arabidopsis thaliana. Following failure of male meiotic cytokinesis in tes mutants, all four microspore nuclei remain within the same cytoplasm, with some completing their developmental programmes to form functional pollen nuclei. Both of the mitotic divisions seen in normal pollen development take place in tes mutants, including the asymmetric division required for the differentiation of gametes; some tes grains perform multiple asymmetric divisions in the same cytoplasm. tes pollen shows a variety of abnormalities subsequent to the cytokinetic defect, including fusion of nuclei, formation of ectopic internal walls, and disruptions to external wall patterning. In addition, ovules fertilized by tes pollen often abort, possibly because of excess paternal genomes in the endosperm. Thus tes mutants not only reveal a gene specific to male meiosis, but aid investigation of a wide range of processes in pollen development and function.

Alleles↗
PubMed2002

Pollen tube development and competitive ability are impaired by disruption of a Shaker K(+) channel in Arabidopsis.

Sexual reproduction in plants requires elongation of the pollen tube through the transmitting tissues toward the ovary. Tube growth rate is a major determinant of pollen competitive ability. We report that a K(+) channel of the Shaker family in Arabidopsis, SPIK, plays an important role in pollen tube development. SPIK was found to be specifically expressed in pollen. When SPIK was heterologously expressed in COS cells, its product formed hyperpolarization-activated K(+) channels. Disruption (T-DNA insertion) of the SPIK coding sequence strongly affected inwardly rectifying K(+)-channel activity in the pollen-grain plasma membrane. Measurements of membrane potential in growing pollen tubes yielded data compatible with a contribution of SPIK to K(+) influx. In vitro pollen germination assays were performed, revealing that the disruption results in impaired pollen tube growth. Analysis of the transmission rate of the disrupted allele in the progeny of heterozygous plants revealed a decrease in pollen competitive ability, the probability of fertilization by mutant pollen being approximately 1.6 times lower than that by wild-type pollen. The whole set of data supports the hypothesis that functional expression of SPIK plays a role in K(+) uptake in the growing pollen tube, and thereby in tube development and pollen competitive ability.

Animals↗
PubMed1995

[An attempt of applying the image processing for the automatic estimation of sampled airborne pollen].

Development of techniques to rapidly and easily estimate an airborne pollen quantity is necessitated in order to make out a appropriate prescription for an allergy patient from medical clinical viewpoints, and in order to research a movement of allergen from medical basic viewpoint. The measurement of airborne pollen quantity required a large labour and time, because the amount of pollen grains is visually measured by naked eye. This paper, as a first step to estimate an airborne pollen quantity full-automatically, discuss techniques to measure the quantity of sampled airborne cedar pollen automatically and rapidly using the image processing techniques. As a result, the following facts are cleared. Automatic measurement is possible to some degree without any special image processing. It is important to eliminate a noise on image as blurs on basefilm for high accuracy measurement. The precision has improved fairly with level correction for image. Sharpening filter is the most appropriate process to improve the accuracy of automatic measurement of sampled airborne pollen. This filtering process has a merit that the operativeness is easy.

Air Pollutants↗
PubMed1987

Developmental strategies of the angiosperm pollen: a biochemical perspective.

The current state of our knowledge of the biochemistry and biochemical cytology of normal pollen development and embryogenic transformation of pollen grains of cultured anthers of angiosperms is reviewed. Recent research shows that normal pollen development is characterized by gene activity for the synthesis of specific mRNAs associated with the gametophytic program. As a result of the trauma of excision and culture of anthers in a mineral salt medium, a small number of the enclosed pollen grains probably synthesize new mRNAs which code for the proteins involved in embryogenic divisions. Since these conclusions are based on the study of a small number of species, the need for sustained investigations on the molecular biology of pollen developmental transformations is emphasized.

Cell Differentiation↗
PubMed1998

Characterization of a desiccation-related protein in lily pollen during development and stress.

This work characterizes a lily (Lilium longiflorum Thunb. cv. Snow Queen) anther (LLA) protein associated with desiccation. Peptide mapping analysis revealed that the abundant LLA-23 doublet contained similar polypeptides, having an isoelectric point of 6.1. Immunoblots of pollen protein from developing anther/pollen confirmed that the LLA-23 protein accumulated only at the later stage of pollen maturation and that the levels remained steady in mature and vital pollen. The accumulation of LLA-23 proteins was correlated with desiccation that naturally occurred in pollen. Subcellular fractionation of pollen proteins revealed that the protein was located in the cytoplasmic fraction. Premature drying of developing pollen confirmed that the concomitant accumulation of LLA-23 was associated with desiccation. Peptide sequence analysis demonstrates similarities between the lily LLA-23 and a family of water-deficit/ripening-induced proteins including LP3 of pine, DS2 of potato, and Asr of tomato and pummelo. In addition, the concomitant accumulation of LLA-23 can be experimentally manipulated by methyl jasmonate (Me-JA) and salicylic acid (SA) as well as by mannitol and methyl viologen. The LLA-23 represents a novel member of the water-deficit/ripening-induced proteins.

Acetates↗
PubMed1997

Fine-scale molecular genetic (RFLP) and physical mapping of a 8.9 cM region on the top arm of Arabidopsis chromosome 5 encompassing the male sterility gene, ms1.

Fine-scale molecular mapping has been conducted using 183 recombinants between the markers lutescens (lu; 17.6 cM) and transparent testa glabra (ttg; 35.5 cM) on the top arm of Arabidopsis thaliana chromosome 5. This region contains a number of genes involved in floral development including Ms1, a gene required for the post-meiotic development of pollen. In homozygous ms1 mutant plants, pollen development is aborted soon after microspore release, regardless of environmental conditions. The ms1 mutation is located at 29.8 +/- 0.8 cM on chromosome 5. Markers have been identified which co-segregate with ms1 and should lie within 39 kb of the gene. The fine-scale map of the lu-ms1-ttg region that has been generated is significantly different from the published integrated map and provides substantially more accurate and higher marker density than the current recombinant inbred map for this region. Using clones derived from four yeast artificial chromosome libraries, a contig has been established between the RFLP markers 4111 and 4556, which encompasses the ms1 gene. This covers a genetic distance of 8.9 cM which corresponds to a physical distance of approximately 1.44 Mb, representing about 1.5-2.0% of the Arabidopsis genome. In this region, 1 cM represents a physical distance of approximately 160 kb.

Arabidopsis↗
PubMed1975

[An analysis of cedar of Lebanon (Cedrus libani A. Rich.) pollen grain development in cultural plantings].

A cytological study of the Cedrus libani mature pollen from 3 culture areas (Italy, France, USSR) has shown that 69-71% of pollen grains have two-celled protallium and antheridial cell. About 5% of pollen grains are characterized by accelerated of delayed development, otherwise apparently normal. The pollen sterility (up to 30% of grains) is due to the abortive spore development. Anomalous cenocyte and multinuclear pollen grains were found thus suggesting that multicellular haploid structures capable of further growth and development may arise in the course of natural anther development.

France↗
PubMed2003

Male-sterility of thermosensitive genic male-sterile rice is associated with premature programmed cell death of the tapetum.

The tapetum plays a crucial role in pollen development. This secretory tissue produces numerous nutritive proteins necessary for pollen maturation. The tapetum, whose cells undergo programmed cell death (PCD), is completely diminished by the time the pollen is fully mature. Our previous studies on a thermosensitive genic male-sterile (TGMS) rice (Oryza sativa L.) suggested that male-sterility was due to failure in pollen development. In this paper we describe how further analysis of the TGMS rice revealed that male-sterility is associated with premature PCD of the tapetum. Cytological observations of TGMS rice anthers at various developmental stages indicated that PCD initiates at an early stage of pollen development and continues until the tapetal cells are completely degraded, resulting in pollen collapse. Transmission electron microscopy showed the morphologically distinct hallmarks of apoptosis, including cytoplasmic shrinkage, membrane blebbing, and vacuolation. Identification of DNA fragmentation using the TUNEL assay supports the hypothesis that premature PCD is associated with male-sterility in the rice. The tissue-specific feature of the thermosensitive genic male-sterile phenotype is discussed with regard to PCD during anther development.

Apoptosis↗
PubMed1998

Identification, sequence analysis and expression studies of novel anther-specific genes of Arabidopsis thaliana.

Relatively little is known about pollen development at the molecular level. For the purpose of gaining understanding of the molecular control of pollen development, a number of Arabidopsis cDNA fragments were isolated using subtractive hybridizations. DNA and RNA hybridizations and sequence analyses indicate that we have isolated cDNAs representing 13 genes. Sequences for 8 of these genes are novel, while those for the remaining 5 genes have substantial similarity to genes previously reported as anther- or pollen-specific. RNA in situ hybridizations with 5 genes revealed that four of them are tapetum-specific with differing temporal expression patterns during pollen development and one is pollen-specific within the flower. Sequence analysis of full-length cDNAs showed that one of the novel genes, ATA7, encodes a protein related to lipid transfer proteins. Another gene, ATA20, encodes a protein with novel repeat sequences and a glycine-rich domain that shares a predicted structure with a known cell wall protein. The full-length ATA27 cDNA encodes a protein similar to the BGL4 beta-glucosidase from Brassica napus. The ATA27 protein is predicted to have an ER retention signal and an acidic isoelectric point, suggesting that it may be localized to the ER lumen. This may be a means of compartmentalization from its substrate(s). Our studies demonstrate that subtractive hybridizations can be used to identify previously unknown genes, which should be valuable tools for further study of pollen and anther development and function.

Amino Acid Sequence↗