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PubMed1999

Expression of the cell cycle in sperm of Arabidopsis: implications for understanding patterns of gametogenesis and fertilization in plants and other eukaryotes.

The relationship between developmental events and the cell cycle was examined in sperm of Arabidopsis thaliana. Sperm of Arabidopsis rapidly enter the S (synthesis) phase of the cell cycle after inception from mitosis of the generative cell. Sperm in pollen grains within anthers continue to synthesize DNA, and at the time of pollination, contain approximately 1.5C DNA. Following pollination, sperm continue through the S phase of the cell cycle during pollen tube growth. By the time pollen tubes reach the ovary, sperm nuclei contain approximately 1.75C DNA. Just prior to double fertilization, sperm nuclei within embryo sacs contain the 2C quantity of DNA. These data indicate that molecular programs associated with the G1-S transition and the S phase of the cell cycle are expressed in sperm cells of developing pollen grains and pollen tubes in Arabidopsis. This pattern of prefertilization S phase activity in the sperm of a flowering plant stands in marked contrast to all other non-plant eukaryotes (from ciliates to yeast to sea urchins to mammals) where sperm remain in G1 during development, prior to the initiation of gametic fusion. In addition, when patterns of cell cycle activity in sperm of Arabidopsis and other flowering plants are compared, developmental analysis reveals that heterochronic alterations (changes in the relative timing of ontogenetic events) in cell cycle activity are a central cause of the diversification of patterns of gametogenesis in higher plants. Finally, comparative analysis of the patterns of cell cycle activity in Arabidopsis and other angiosperms may be used to predict which flowering plants will be amenable to development of successful in vitro fertilization techniques.

Arabidopsis↗
PubMed2000

Isolation and characterization of cDNAs expressed in the early stages of flavonol-induced pollen germination in petunia.

Petunia (Petunia hybrida) pollen requires flavonols (Fl) to germinate. Adding kaempferol to Fl-deficient pollen causes rapid and synchronous germination and tube outgrowth. We exploited this system to identify genes responsive to Fls and to examine the changes in gene expression that occur during the first 0.5 h of pollen germination. We used a subtracted library and differential screening to identify 22 petunia germinating pollen clones. All but two were expressed exclusively in pollen and half of the clones were rare or low abundance cDNAs. RNA gel-blot analysis showed that the steady-state transcript levels of all the clones were increased in response to kaempferol. The sequences showing the greatest response to kaempferol encode proteins that have regulatory or signaling functions and include S/D4, a leucine-rich repeat protein, S/D1, a LIM-domain protein, and D14, a putative Zn finger protein with a heme-binding site. Eight of the clones were novel including S/D10, a cDNA only expressed very late in pollen development and highly up-regulated during the first 0.5 h of germination. The translation product of the S/D3 cDNA shares some features with a neuropeptide that regulates guidance and growth in the tips of extending axons. This study confirmed that the bulk of pollen mRNA accumulates well before germination, but that specific sequences are transcribed during the earliest moments of Fl-induced pollen germination.

Amino Acid Sequence↗
PubMed1994

dsRNA degrading nucleases are differentially expressed in tobacco anthers.

Nucleases, capable of digesting double-stranded RNAs are mainly confined to extracellular fractions of tobacco anthers and diffusate of mature pollen. dsRNAse activity is about 150-fold higher in anther fractions than in crude nuclease extracts from tobacco leaves. The level of dsRNAse activity varies during pollen development from the microspore stage to maturity. In the anther soluble fraction, dsRNAse activity reached a maximum (approx. 50 units/anther) at the end of microspore mitosis and then decreased continuously until the stage of almost mature anthers. In contrast, the nuclease activity associated with pollen increased continuously reaching a maximum (5 units/anther), during subsequent stages of pollen maturation. Gel electrophoretic analysis revealed four slowly migrating sugar-unspecific nucleases (active against DNA and RNA) and three faster migrating RNases which were all able to digest dsRNA. Competition experiments showed that the sugar-unspecific nucleases accounted for 95% of the total dsRNAse activity. Anther extracellular nucleases were further characterized after partial purification on NADP-agarose: dsRNAse activity had a pH optimum at 5.5, was strongly inhibited by NaCl and by 1 mM Zn2+ and was insensitive to EDTA which could stimulate activity in crude preparations. Analysis of the activity with defined substrates showed that ssRNA is more readily degraded than dsRNA and that both, endo- and exonucleolytic activities are detected.

Centrifugation, Density Gradient↗
PubMed1985

Initiation of Postmeiotic beta-Galactosidase Synthesis during Microsporogenesis in Oilseed Rape.

The synthesis of beta-galactosidase during Brassica campestris pollen development results from the transcription of the haploid genome. A quantitative cytochemical method has been developed in which 5-bromo-4-chloro-3-indoxyl-beta-d-galactoside is used as substrate giving a blue-green final reaction product. We have recently detected oilseed rape plants which are heterozygous for the beta-galactosidase locus, in which 50% of the pollen grains produced are Gal (having enzyme activity), while the other 50% are gal (enzyme deficient). The gal pollen grains served as a built-in control during microspectrophotometric determinations of enzyme activity. The present study has identified the developmental phase at which synthesis of the enzyme commenced. Activity is absent in microsporocytes, tetrads, and at microspore release. Enzyme activity is first detected in the young microspores and, by early vacuolate period, there is an increase in the rate of enzyme activity. A second period of increased enzyme synthesis occurred prior to generative cell division, although the rate is reduced in mature pollen.

Journal Article↗
PubMed1981

Ontogeny of the barley plant as related to mutation expression and detection of pollen mutations.

Clustering of mutant pollen grains in a population of normal pollen due to premeiotic mutational events complicates translating mutation frequencies into rates. Embryo ontogeny in barley will be described and used to illustrate the formation of such mutant clusters. The nature of the statistics for mutation frequency will be described from a study of the reversion frequencies of various waxy mutants in barley. Computer analysis by a "jackknife" method of the reversion frequencies of a waxy mutant treated with the mutagen sodium azide showed a significantly higher reversion frequency than untreated material. Problems of the computer analysis suggest a better experimental design for pollen mutation experiments. Preliminary work on computer modeling for pollen development and mutation will be described.

Computers↗
PubMed2000

Purification of a jojoba embryo fatty acyl-coenzyme A reductase and expression of its cDNA in high erucic acid rapeseed.

The jojoba (Simmondsia chinensis) plant produces esters of long-chain alcohols and fatty acids (waxes) as a seed lipid energy reserve. This is in contrast to the triglycerides found in seeds of other plants. We purified an alcohol-forming fatty acyl-coenzyme A reductase (FAR) from developing embryos and cloned the cDNA encoding the enzyme. Expression of a cDNA in Escherichia coli confers FAR activity upon those cells and results in the accumulation of fatty alcohols. The FAR sequence shows significant homology to an Arabidopsis protein of unknown function that is essential for pollen development. When the jojoba FAR cDNA is expressed in embryos of Brassica napus, long-chain alcohols can be detected in transmethylated seed oils. Resynthesis of the gene to reduce its A plus T content resulted in increased levels of alcohol production. In addition to free alcohols, novel wax esters were detected in the transgenic seed oils. In vitro assays revealed that B. napus embryos have an endogenous fatty acyl-coenzyme A: fatty alcohol acyl-transferase activity that could account for this wax synthesis. Thus, introduction of a single cDNA into B. napus results in a redirection of a portion of seed oil synthesis from triglycerides to waxes.

Aldehyde Oxidoreductases↗
PubMed2000

Cloning of cDNA encoding NtEPc, a marker protein for the embryogenic dedifferentiation of immature tobacco pollen grains cultured in vitro.

We partially purified three Nicotiana tabacum L. embryogenic pollen-abundant phosphoproteins (NtEPa to c) which appeared in the cells undergoing a dedifferentiation process from immature pollen grains to embryogenic cells, caused by glutamine-deficiency in vitro. All the NtEPs had a highly conserved N-terminal amino acid sequence. Using degenerate oligonucleotide probes designed from the amino acid sequences, the cDNA for NtEPc was isolated from a cDNA library of pollen cultured in glutamine-free medium The cDNA sequence showed moderate homology with several type-1 copper-binding glycoproteins and with a kind of early nodulin though its function could not be predicted. Expression analysis revealed that the level of mRNA for NtEPc was high during the dedifferentiation and also in the very early period of pollen embryogenesis but it was low in the developmental process of microspores/pollen in anthers, in the in vitro maturation process and both in the stational and logarithmic growth phases of tobacco BY-2 cells. Furthermore, an acidic medium pH, which promoted the induction of dedifferentiation increased the level of mRNA for NtEPc, whereas the presence of 6-benzylaminopurine, which inhibited it, decreased the level. These results suggest that the expression of NtEPc gene is correlated with the dedifferentiation but not with pollen development or cell division.

Amino Acid Sequence↗
PubMed2002

A conditionally fertile coi1 allele indicates cross-talk between plant hormone signalling pathways in Arabidopsis thaliana seeds and young seedlings.

Jasmonates (JAs) regulate Arabidopsis thaliana (L.) Heynh. wound and defense responses, pollen development, and stress-related growth inhibition. Significantly, each of these responses requires COI1, an F-box protein. We fused firefly luciferase as a reporter to the JA-responsive promoter for the vegetative storage protein gene (VSP) and used this to screen for mutants that failed to express luciferase in the presence of JA, isolating a mutant designated coi1-16. Comparisons with coi1-1 and jar1-1 plants indicated that coi1-16 was only slightly more sensitive to JA than coi1-1 plants. However, whilst coi1-16 plants failed to produce viable pollen at 22 degrees C, they were fertile at 16 degrees C. Therefore, unlike the other coi1 mutants, coi1-16 could be maintained as a pure line and did not require selection. We have used coi1-16 seeds to define novel interactions between JA and other hormone signalling pathways in seed germination and in the development of young seedlings.

Abscisic Acid↗
PubMedDate not supplied

MAPKs entry into the nucleus at specific interchromatin domains in plant differentiation and proliferation processes.

Mitogen-activated protein kinases (MAPKs) are involved in the signaling of extracellular stimuli in eukaryotes, including plants. Different MAPKs have recently been shown to be expressed during plant cell proliferation and developmental processes such as pollen development and embryogenesis, but the structural subdomain where these MAPKs are targeted in the nucleus has not yet been characterized. We have determined the changes in the expression and subcellular localization of ERK homologues, proteins belonging to the MAPK family, and MAPK-active forms in two plant developmental processes which involved differentiation (pollen maturation) and proliferation (the initials of pollen embryogenesis). Immunofluorescence and immunogold labeling in the species studied showed that the progression of differentiation and proliferation was accompanied by an increase in the expression of ERKs and MAPK activation together with a translocation to the nucleus. Combining ultrastructural cytochemistry and immunogold for RNA and phosphorylated proteins we have identified the nuclear sites housing these MAPKs in areas of the interchromatin region enriched in RNA and phosphoproteins that include clusters of interchromatin granules. This could suggest a role of these MAPKs in the early events of activation of the transcription and processing machinery, via phosphorylation, which subsequently would be recruited to the transcription sites. The association of the nuclear localization of MAPKs with the progression through the cell cycle and the commitment toward differentiation in the two plant developmental processes can be correlated.

Cell Differentiation↗
PubMed2000

The Arabidopsis male-sterile mutant, opr3, lacks the 12-oxophytodienoic acid reductase required for jasmonate synthesis.

Jasmonic acid (JA) and its precursor 12-oxophytodienoic acid (OPDA) act as plant growth regulators and mediate responses to environmental cues. To investigate the role of these oxylipins in anther and pollen development, we characterized a T-DNA-tagged, male-sterile mutant of Arabidopsis, opr3. The opr3 mutant plants are sterile but can be rendered fertile by exogenous JA but not by OPDA. Cloning of the mutant locus indicates that it encodes an isozyme of 12-oxophytodienoate reductase, designated OPR3. All of the defects in opr3 are alleviated by transformation of the mutant with an OPR3 cDNA. Our results indicate that JA and not OPDA is the signaling molecule that induces and coordinates the elongation of the anther filament, the opening of the stomium at anthesis, and the production of viable pollen. Just as importantly, our data demonstrate that OPR3 is the only isoform of OPR capable of reducing the correct stereoisomer of OPDA to produce JA required for male gametophyte development.

Amino Acid Sequence↗
PubMed1992

Isolation of genes abundantly expressed in rice anthers at the microspore stage.

A cDNA library of rice (Oryza sativa L.) has been constructed from anthers at an early stage of pollen development. By differential screening of the library, we have isolated cDNAs of two genes, designated as Osc4 and Osc6, that are abundantly expressed in anthers containing tetrads and uninucleate microspores, but are not expressed in leaves or roots. Expression of Osc4 is absent in mature anthers, while Osc6 is present although the expression decays during pollen maturation. A comparison of the nucleotide and deduced amino acid sequences with those in data banks has not shown significant homology to known molecules.

Amino Acid Sequence↗
PubMed2003

Immunohistochemical studies on translocation of pollen S-haplotype determinant in self-incompatibility of Brassica rapa.

The self-incompatibility system in Brassica is controlled by the S-locus, which contains S-receptor kinase (SRK) and S-locus protein 11 (SP11). SRK and SP11 control stigma and pollen S-haplotype specificity, respectively. SP11 binding to SRK induces the autophosphorylation of SRK, which triggers the signaling cascade that results in the rejection of self-pollen. The localization of SP11 protein during pollen development and pollination, however, have never been demonstrated. In this study, we examined the localization of S(8)-SP11 protein in the anther or pollinated stigma by immuno-electron microscopy. The immunostaining suggested that S(8)-SP11 was secreted from the tapetal cell into the anther locule as a cluster and translocated to the pollen surface at the early developmental stage of the anther. During the pollination process, SP11 was translocated from the pollen surface to the papilla cell, and then penetrated the cuticle layer of the papilla cell to diffuse across the pectin cellulose layer. Furthermore, SP11 protein could only penetrate the cuticle layer of the papilla cell in the presence of pollen grains, and could not penetrate on its own. This suggests that another factor from the pollen grain is needed for SP11 protein to penetrate the papilla cell wall.

Biological Transport↗
PubMed1992

Isolation and characterization of a tobacco gene with homology to pectate lyase which is specifically expressed during microsporogenesis.

A genomic clone has been isolated which contains an open reading frame of 1191 bp interrupted by two small introns. The ORF has been sequenced and the transcriptional start determined. The predicted amino acid sequence shows homology to the deduced amino acid sequences of two pollen-specific pectate lyase genes identified in tomato. The genomic clone was isolated using a partial cDNA clone, TP10, which had been isolated from a Nicotiana tabacum pollen cDNA library by means of differential screening. TP10 has been fully sequenced and contains an open reading frame of 792 bp which shows 96% homology to the ORF in the genomic clone. The transcript corresponding to TP10 is maximally expressed late in pollen development, and has not been detected in vegetative tissues.

Amino Acid Sequence↗
PubMed2004

Cloning and expression pattern of a novel microspore-specific gene encoding hypersensitive-induced response protein (LjHIR1) from the model legume, Lotus japonicus.

In order to understand the microspore and pollen development, recently, we have isolated a number of anther-specific genes in the model legume, Lotus japonicus. From these anther-specific genes, we identified one novel microspore-specific gene, LjImfb-c82. In order to determine the molecular characterization of LjImfb-c82, full-length cDNA clone was first isolated and sequenced. It encoded a protein of 286 amino acids (LjHIR1), which had sequence similarity to Hypersensitive-Induced Response like protein. LjHIR1 was specifically expressed in microspore on the in situ hybridization experiment. From the sequence similarity to prohibitin-domain protein, the LjHIR1 might be related to ion channel regulation in microspore development.

Amino Acid Sequence↗
PubMed1991

Characterization of pollen polygalacturonase encoded by several cDNA clones in maize.

A full-length cDNA clone, named PG1, abundantly expressed in late stages of pollen development, has been isolated from a cDNA library using a differential screening method with cDNA probes representative of microspores at early or late developmental stages. The encoded 410 amino acid polypeptide has significant homology with various polygalacturonases (PG) described elsewhere. Two polypeptides, of 49 and 53 kDa respectively, have been identified in the active PG fraction, isolated from mature pollen by immuno-cross-reaction with tomato PG antibodies. According to their N-terminal sequence, they can be identified as being mature peptides encoded by the PG1 cDNA clone. We propose that these two proteins derive from a unique precursor through several post-translational events, including the excision of a 22 amino-terminal signal peptide and glycosylation. PG-encoding genes from a small genomic family. Sequence analysis of three PG cDNA clones shows that they are closely related. The divergence of nucleotides between these three cDNA clones is 1%. They encode the same product.

Amino Acid Sequence↗
PubMed2005

Arabidopsis SOMATIC EMBRYOGENESIS RECEPTOR KINASES1 and 2 are essential for tapetum development and microspore maturation.

Among the >200 members of the leucine-rich repeat receptor kinase family in Arabidopsis thaliana, only a few have been functionally characterized. Here, we report a critical function in anther development for the SOMATIC EMBRYOGENESIS RECEPTOR KINASE1 (SERK1) and SERK2 genes. Both SERK1 and SERK2 are expressed widely in locules until stage 6 anthers and are more concentrated in the tapetal cell layer later. Whereas serk1 and serk2 single insertion mutants did not show developmental phenotypes, serk1 serk2 double mutants were not able to produce seeds because of a lack of pollen development in mutant anthers. In young buds, double mutant anthers developed normally, but serk1 serk2 microsporangia produced more sporogenous cells that were unable to develop beyond meiosis. Furthermore, serk1 serk2 double mutants developed only three cell layers surrounding the sporogenous cell mass, whereas wild-type anthers developed four cell layers. Further confocal microscopic and molecular analyses showed that serk1 serk2 double mutant anthers lack development of the tapetal cell layer, which accounts for the microspore abortion and male sterility. Taken together, these findings demonstrate that the SERK1 and SERK2 receptor kinases function redundantly as an important control point for sporophytic development controlling male gametophyte production.

Arabidopsis↗
PubMed2006

[BcMF4 gene, encoding a leucine-rich repeat protein, plays a role in male fertility in Chinese cabbage-pak-choi].

The BcMF4 (Brassica campestris Male Fertility 4) gene was previously isolated from the fertile B line of Chinese cabbage-pak-choi (Brassica campestris ssp. chinensis var. communis, syn. B. rapa ssp. chinensis var. communis). In the present paper, based on the cDNA sequence of BcMF4, primers were designed and used to amplify two fragments from the cDNA of flower buds of Chinese cabbage-pak-choi. Two produced fragments were introduced separately into binary vector pBI121 in antisense and sense orientations. The generated RNA interference (RNAi) vector was then mobilized into Agrobacterium tumefaciens strain LBA4404. The A. tumefaciens harboring the BcMF4 fragments was transformed to flowering Chinese cabbage (B. campestris ssp. chinensis var. parachinensis) via tissue culture. Approximately 45.8% of the pollen grains from 72.2% of RNAi plants exhibited abnormal in their shapes, and only 23.7% of the pollen grains from these plants germinated normally. Northern blotting demonstrated that the phenotypic change of pollen grains resulted from the inhibition of expression of the BcMF4 due to the insertion of the transgene. This indicates that functional interrupting of BcMF4 by RNAi resulted in partial pollen abortion in flowering Chinese cabbage, suggesting that the product of BcMF4 gene plays an important role during pollen development of Chinese cabbage such as Chinese cabbage-pak-choi and flowering Chinese cabbage.

Brassica↗
PubMed2001

Oxytropism: a new twist in pollen tube orientation.

Chemical gradients and structural features within the pistil have been previously proposed as factors determining the directionality of pollen tube growth. In this study, we examine the behavior of pollen of eight species germinated in a dynamic oxygen gradient. While the germination rates of some species decreased directly with decreasing oxygen tension, other species showed no decrease in germination at oxygen tensions as low as 2 kPa. In one species, germination was consistently greater at decreased oxygen tensions than at ambient atmospheric levels. In three of the eight species tested, the developing pollen tube showed clear directional growth away from the more-oxygenated regions of the growth medium, while in one species growth was towards the more-oxygenated region. The remaining four species showed random tube growth. The pattern of oxytropic responses among the taxa suggests that this tropic behavior is both widespread and phylogenetically unpredictable.

Germination↗