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PubMed1997

Analysis of microspore-specific promoters in transgenic tobacco.

In order to modify the early stages of pollen development in a transgenic context microspore-specific promoters are required. We tested two putatively microspore-specific promoters, the Bp4 promoter from rapeseed and the NTM19 promoter from tobacco. Expression of the gus and barnase reporter genes under the control of these two promoters was studied in transgenic tobacco. Contrary to expectations, the Bp4 promoter became active only after the first pollen mitosis, and not in the microspores. The NTM19 promoter turned out to be highly microspore-specific and directed very high levels of gus expression to the unicellular microspores. The NTM19-barnase transgene caused cell-autonomous death at the mid-unicellular microspore stage, whereas Bp4-barnase induced cell ablation of early to mid-bicellular pollen. Both promoter-barnase transgenes did not affect the sporophyte and were inherited through the female germline. These results show that both the NTM19 and Bp4 promoters are expressed only in the male germline, and that the NTM19 promoter is an excellent tool to direct high levels of transgene expression exclusively to the microspores. This may have important biotechnological applications.

Bacterial Proteins↗
PubMed2002

Reduction in amounts of mitochondrial DNA in the sperm cells as a mechanism for maternal inheritance in Hordeum vulgare.

It is known that extranuclear organelle DNA is inherited maternally in the majority of angiosperms. The mechanisms for maternal inheritance have been well studied in plastids but not in mitochondria. In the present study we examined the mitochondrial DNA in the male reproductive cells of Hordeum vulgare L. by immunoelectron microscopy. Our results show that the number of anti-DNA gold particles on sections of sperm cell mitochondria decreased by 97% during pollen development. The reduction occurred rapidly in the generative cells and subsequently in the sperm cells, concomitant with a remarkable reduction in mitochondrial volume. It seems that the copy numbers of mitochondrial DNA were reduced in the male reproductive cells, which may be a possible mechanism by which paternal transmission is inhibited. Unlike mitochondria, plastids are excluded from the generative cells during the first pollen mitosis. These data suggest a mechanism for maternal inheritance of mitochondria in angiosperms and for independent control of inheritance of mitochondria and plastids in H. vulgare.

DNA, Mitochondrial↗
PubMed1997

The Arabidopsis MALE STERILITY 2 protein shares similarity with reductases in elongation/condensation complexes.

The Arabidopsis thaliana MALE STERILITY 2 (MS2) gene product is involved in male gametogenesis. The first abnormalities in pollen development of ms2 mutants are seen at the stage in microsporogenesis when microspores are released from tetrads. Expression of the MS2 gene is observed in tapetum of wild-type flowers at, and shortly after, the release of microspores from tetrads. The MS2 promoter controls GUS expression at a comparable stage in the tapetum of transgenic tobacco containing an MS2 promoter-GUS fusion. The occasional pollen grains produced by mutant ms2 plants have very thin pollen walls. They are also sensitive to acetolysis treatment, which is a test for the presence of an exine layer. The MS2 gene product shows sequence similarity to a jojoba protein that converts wax fatty acids to fatty alcohols. A possible function of the MS2 protein as a fatty acyl reductase in the formation of pollen wall substances is discussed.

Amino Acid Sequence↗
PubMed2005

Androgenic switch: an example of plant embryogenesis from the male gametophyte perspective.

Embryogenesis in plants is a unique process in the sense that it can be initiated from a wide range of cells other than the zygote. Upon stress, microspores or young pollen grains can be switched from their normal pollen development towards an embryogenic pathway, a process called androgenesis. Androgenesis represents an important tool for research in plant genetics and breeding, since androgenic embryos can germinate into completely homozygous, double haploid plants. From a developmental point of view, androgenesis is a rewarding system for understanding the process of embryo formation from single, haploid microspores. Androgenic development can be divided into three main characteristic phases: acquisition of embryogenic potential, initiation of cell divisions, and pattern formation. The aim of this review is to provide an overview of the main cellular and molecular events that characterize these three commitment phases. Molecular approaches such as differential screening and cDNA array have been successfully employed in the characterization of the spatiotemporal changes in gene expression during androgenesis. These results suggest that the activation of key regulators of embryogenesis, such as the BABY BOOM transcription factor, is preceded by the stress-induced reprogramming of cellular metabolism. Reprogramming of cellular metabolism includes the repression of gene expression related to starch biosynthesis and the induction of proteolytic genes (e.g. components of the 26S proteasome, metalloprotease, cysteine, and aspartic proteases) and stress-related proteins (e.g. GST, HSP, BI-1, ADH). The combination of cell tracking systems with biochemical markers has allowed the key switches in the developmental pathway of microspores to be determined, as well as programmed cell death to be identified as a feature of successful androgenic embryo development. The mechanisms of androgenesis induction and embryo formation are discussed, in relation to other biological systems, in special zygotic and somatic embryogenesis.

Apoptosis↗
PubMed1973

The Nutritional Role of Pistil Exudate in Pollen Tube Wall Formation in Lilium longiflorum: II. Production and Utilization of Exudate from Stigma and Stylar Canal.

Detached pistils of Lilium longiflorum were labeled with d-glucose-U-(14)C 24 hours after anthesis and then sampled for the next 6 days to determine the appearance of label into exudate from the stylar canal and the stigmatic surface of the pistil. Results were obtained with unpollinated cv. Ace pistils and pollinated cv. Ace pistils, selfed or crossed (cv. Croft pollen). Limited data were also obtained on cv. Croft pistils, selfed or crossed (cv. Ace pollen).Exudate appeared in the canal and on the stigmatic surface soon after anthesis. In unpollinated pistils it continued to accumulate for about 5 days in the canal and for the full term of the experiment, 7 days, on the stigmatic surface. Canal exudate eventually mixed with stigmatic exudate in unpollinated pistils. Pollination interrupted the flow of exudate, and a portion of the pistil secretion product was diverted toward the synthetic requirements of the developing pollen tubes. Two days after pollination, the specific radioactivity of pollen tube cytoplasm had reached a level comparable to canal exudate. Twenty-four hours later, the specific radioactivity of pollen tube wall substance was about 80% of the value found in canal exudate. Similar patterns of (14)C incorporation and similar carbohydrate contents were obtained from self- and cross-pollinated pistils, although the former contained pollen tubes of less than one-half the length of the latter.

Journal Article↗
PubMed2003

Functional characterization and expression analyses of the glucose-specific AtSTP9 monosaccharide transporter in pollen of Arabidopsis.

A genomic clone and the corresponding cDNA of a new Arabidopsis monosaccharide transporter AtSTP9 were isolated. Transport analysis of the expressed protein in yeast showed that AtSTP9 is an energy-dependent, uncoupler-sensitive, high-affinity monosaccharide transporter with a K(m) for glucose in the micromolar range. In contrast to all previously characterized monosaccharide transporters, AtSTP9 shows an unusual specificity for glucose. Reverse transcriptase-polymerase chain reaction analyses revealed that AtSTP9 is exclusively expressed in flowers, and a more detailed approach using AtSTP9 promoter/reporter plants clearly showed that AtSTP9 expression is restricted to the male gametophyte. AtSTP9 expression is not found in other floral organs or vegetative tissues. Further localization on the cellular level using a specific antibody revealed that in contrast to the early accumulation of AtSTP9 transcripts in young pollen, the AtSTP9 protein is only found weakly in mature pollen but is most prominent in germinating pollen tubes. This preloading of pollen with mRNAs has been described for genes that are essential for pollen germination and/or pollen tube growth. The pollen-specific expression found for AtSTP9 is also observed for other sugar transporters and indicates that pollen development and germination require a highly regulated supply of sugars.

Amino Acid Sequence↗
PubMed2001

Isolation and characterization of an endonuclease synthesized by barley (Hordeum vulgare L.) uninucleate microspores.

Few biochemical and molecular details are available on microspore growth and development. In this work, a nuclease was partially purified from diffusates of barley (Hordeum vulgare L.) microspores by using concanavalin-A as ligand. The chromatographic preparation contained a 34-kDa protein with nucleolytic activity; the enzyme (called BMN: barley microspore nuclease) was very stable at pH > 8.0 and temperatures below 50 degrees C. Activity was highest at pH 5.6 and increased almost exponentially with temperature until a breakpoint between activity and stability was reached at 70 degrees C. Although BMN was able to cleave RNA, the enzyme showed a remarkable preference for DNA, especially in the single-stranded form. The best homopolymeric substrates were poly(dA) and poly(A), whereas poly(dC), poly(G) and poly(I) were almost completely uncleaved. When incubated with intact nuclei, BMN caused a nucleosomal DNA ladder of approximately 200 bp. On the basis of DNA laddering, substrate specificity, Mg2+ -dependence and best performance at apoplastic pH, BMN can be referred to as a putative apoptotic nuclease involved in pollen development.

Apoptosis↗
PubMed1994

Expression of the BnmNAP subfamily of napin genes coincides with the induction of Brassica microspore embryogenesis.

Brassica napus cv. Topas microspores can be diverted from pollen development toward haploid embryo formation in culture by subjecting them to a heat stress treatment. We show that this switch in developmental pathways is accompanied by the induction of high levels of napin seed storage protein gene expression. Changes in the plant growth or microspore culture conditions were not by themselves sufficient to induce napin gene expression. Specific members of the napin multigene family were cloned from a cDNA library prepared from microspores that had been induced to undergo embryogenesis. The majority of napin clones represented three members (BnmNAP2, BnmNAP3 and BnmNAP4) that, along with a previously isolated napin genomic clone (BngNAP1), constitute the highly conserved BnmNAP subfamily of napin genes. Both RNA gel blot analysis, using a subfamily-specific probe, and histochemical analysis of transgenic plants expressing a BngNAP1 promoter-beta-glucuronidase gene fusion demonstrated that the BnmNAP subfamily is expressed in embryogenic microspores as well as during subsequent stages of microsporic embryo development.

2S Albumins, Plant↗
PubMedDate not supplied

High-pressure freeze fixation reveals novel features during ontogenesis of the vegetative cell in Ledebouria pollen: an ultrastructural and cytochemical study.

The ultrastructure of the vegetative cell in the pollen of Ledebouria socialis Roth (Hyacinthaceae) was investigated from microspore mitosis to anthesis. As a result of the good preservation quality achieved with high-pressure freeze fixation and freeze substitution, novel structural features were observed. Extensive endomembrane compartments emerging at the onset of lipid and starch mobilization, were identified as protein bodies by using video-enhanced contrast light microscopy. Thus, proteins, apart from starch and lipids, represent a third class of important intermediary storage substances in developing pollen. The close spatial relationship between protein bodies, endoplasmic reticulum (ER), and storage lipids suggest that protein bodies and ER contribute to lipid digestion. Immediately prior to anthesis the protein bodies become transformed into unspecialized vacuoles as a result of the gradual dissolution of their contents; the formation of the protein bodies remains still to be elucidated. The ER proliferates extensively during pollen ontogenesis, thereby changing its ultrastructure and spatial organization. Microfilaments were detected during all developmental stages, in particular microtubule-associated single microfilaments. The microfilaments are likely to be composed of actin as shown by immunogold labeling.

Actin Cytoskeleton↗
PubMed1991

Plant mitochondrial mutations and male sterility.

A characteristic of CMS mutations in plants, in contrast to the single base changes in human mitochondrial mutants (49, 140), is the presence of chimeric genes or chimeric loci; different open reading frames are joined together, or placed in proximal locations and cotranscribed with standard mitochondrial genes. Despite much progress, and the identification of several mitochondrial loci that specify CMS, the molecular basis of this defect is not understood in any plant species. Observations of altered electron transport in Petunia and toxin-mediated membrane disruption in maize plants, bacteria, and yeast expressing the maize urf13 gene product, provide clues to possible mechanisms for disruption of pollen development. Whether disruption in a particular mitochondrial function is at the root of CMS in all species, or whether defects in numerous mitochondrial activities can produce sterility, will only be revealed by further probing of physiological and biochemical defects present in CMS genotypes.

Agriculture↗
PubMed1998

A maize pectin methylesterase-like gene, ZmC5, specifically expressed in pollen.

Pectin methylesterase (PME) is responsible for the demethylation of pectin prior to pectin's degradation by the combined activities of polygalacturonase and pectate lyase. We have differentially screened a maize pollen cDNA library to detect cDNA clones whose genes are specifically expressed in pollen. One group of clones resulting from this screen showed similarity (between 18% and 41% identity) with plant and fungal PMEs. The full-length clone from this group, ZmC5, identifies a small gene family (at least 2 members) when used as a probe on genomic Southern blots. Northern analysis reveals that the ZmC5 transcript is expressed specifically in late pollen development. This tissue-specific gene expression programme is further confirmed in transgenic tobacco plants harbouring ZmC5 promoter/GUS chimeric gene constructs.

Base Sequence↗
PubMed1996

The rf2 nuclear restorer gene of male-sterile T-cytoplasm maize.

The T cytoplasm of maize serves as a model for the nuclear restoration of cytoplasmic male sterility. The rf2 gene, one of two nuclear genes required for fertility restoration in male-sterile T-cytoplasm (cmsT) maize, was cloned. The protein predicted by the rf2 sequence is a putative aldehyde dehydrogenase, which suggests several mechanisms that might explain Rf2-mediated fertility restoration in cmsT maize. Aldehyde dehydrogenase may be involved in the detoxification of acetaldehyde produced by ethanolic fermentation during pollen development, may play a role in energy metabolism, or may interact with URF13, the mitochondrial protein associated with male sterility in cmsT maize.

Acetaldehyde↗
PubMed1997

A strong inhibitor of gene expression in the 5' untranslated region of the pollen-specific LAT59 gene to tomato.

Promoter sequences that direct pollen-specific expression have been previously identified in the LAT59 (for late anther tomato) gene. Here, we show that the LAT59 sequences encoding the 5' untranslated region inhibit expression of reporter genes by > 20-fold in transient expression experiments and up to 300-fold after stable transformation. Inhibition occurred in somatic cells as well as in pollen. Our results indicate that the inhibitor still functions after pollen germination and therefore does not modulate the level of the LAT59 protein during pollen development. The presence of the leader sequence dramatically decreased mRNA accumulation but without affecting translation rate and mRNA stability. We believe that the leader inhibits transcription. We mapped the inhibitor to a region in the leader that coincides with a putative stem-loop and present evidence that this stem-loop participates in inhibition.

Gene Expression Regulation, Plant↗
PubMed2005

Potential cytoplasmic inheritance in Wisteria sinensis and Robinia pseudoacacia (Leguminosae).

We examined pollen cells of Wisteria sinensis and Robinia pseudoacacia (Leguminosae) to determine a possible mode for cytoplasmic inheritance in these species. Epifluorescence microscopy revealed distinct mature generative cells. Mature generative cells of W. sinensis were associated with large numbers of punctuated fluorescent signals corresponding to cytoplasmic DNA aggregates, but no fluorescent signals were observed in the generative cells of R. pseudoacacia. Closer examination showed that the punctate fluorescent signals corresponded to plastid but not mitochondrial DNA. These results suggest a strong potential for paternal transmission of the plastid genome in W. sinensis. Electron microscopy confirmed the presence of plastids in the generative cells of W. sinensis and the absence of plastids in R. pseudoacacia cells due to an unequal distribution of plastids during the first pollen mitosis. Mitochondria were present and intact in the mature generative cells of both species. The lack of fluoresced mitochondrial DNA suggests a very low level of mitochondrial DNA in the cells. Immunoelectron microscopy demonstrated that the labeling of mitochondrial DNA in these cells was reduced by nearly 90% during pollen development. Such a dramatic reduction suggests an active degradation of paternal mitochondrial DNA, which may contribute greatly to the maternal inheritance of mitochondria. In short, we found that W. sinensis exhibits a strong potential for paternal transmission of plastids and that both W. sinensis and R. pseudoacacia appear to share the same mechanism for maternal mitochondrial inheritance.

Cytoplasm↗
PubMed2000

The dyad gene is required for progression through female meiosis in Arabidopsis.

In higher plants the gametophyte consists of a gamete in association with a small number of haploid cells, specialized for sexual reproduction. The female gametophyte or embryo sac, is contained within the ovule and develops from a single cell, the megaspore which is formed by meiosis of the megaspore mother cell. The dyad mutant of Arabidopsis, described herein, represents a novel class among female sterile mutants in plants. dyad ovules contain two large cells in place of an embryo sac. The two cells represent the products of a single division of the megaspore mother cell followed by an arrest in further development of the megaspore. We addressed the question of whether the division of the megaspore mother cell in the mutant was meiotic or mitotic by examining the expression of two markers that are normally expressed in the megaspore mother cell during meiosis. Our observations indicate that in dyad, the megaspore mother cell enters but fails to complete meiosis, arresting at the end of meiosis 1 in the majority of ovules. This was corroborated by a direct observation of chromosome segregation during division of the megaspore mother cell, showing that the division is a reductional and not an equational one. In a minority of dyad ovules, the megaspore mother cell does not divide. Pollen development and male fertility in the mutant is normal, as is the rest of the ovule that surrounds the female gametophyte. The embryo sac is also shown to have an influence on the nucellus in wild type. The dyad mutation therefore specifically affects a function that is required in the female germ cell precursor for meiosis. The identification and analysis of mutants specifically affecting female meiosis is an initial step in understanding the molecular mechanisms underlying early events in the pathway of female reproductive development.

Arabidopsis↗
PubMed2000

Genes normally expressed in the endosperm are expressed at early stages of microspore embryogenesis in maize.

Reproduction in flowering plants is characterized by double fertilization and the resulting formation of both the zygotic embryo and the associated endosperm. In many species it is possible to experimentally deviate pollen development towards an embryogenic pathway. This developmental switch, referred to as microspore embryogenesis or androgenesis, leads to the formation of embryos similar to zygotic embryos. In a screen for genes specifically expressed during early androgenesis, two maize genes were isolated by mRNA differential display. Both genes represent new molecular markers expressed at a very young stage of androgenic embryogenesis. When their expression pattern was studied during normal reproductive development, both showed early endosperm-specific expression. Investigation of the cytological features of young androgenic embryos revealed that they present a partially coenocytic organization similar to that of early endosperm. These findings suggest that maize androgenesis may possibly involve both embryogenesis and the establishment of endosperm-like components.

Blotting, Southern↗
PubMed1994

Characterization of a tobacco gene encoding a pollen-specific polygalacturonase.

We report here the isolation and characterization of a gene which is specifically expressed during late pollen development in Nicotiana tabacum L. cv. Havana and which exhibits homology to bacterial, fungal and plant polygalacturonases. This gene is ca. 4.3 kb, from the transcription start-site to the 3' polyadenylation-site sequences. It contains three introns of 620, 706 and 1400 bp and encodes a 1.5 kb message that contains an A-rich 5'-untranslated-leader sequence of 81 bases and a variable-length 3'-untranslated sequence of between 180 and 320 bases. Located within intron 3 is a 414 bp sequence which exhibits 79% homology to a sequence within the endochitinase gene; both sequences share the same internal repeat structure and exhibit features consistent with them being defective transposable elements. The predicted protein sequence coded for by Npg1 shows, in addition to a number of highly conserved cysteines, four conserved domains with the bacterial and fungal polygalacturonase genes. The pollen-specific polygalacturonases as a group can be distinguished from the fruit-ripening polygalacturonases by a number of criteria. It is suggested that these differences reflect the functional differences between plant endo- and exo-polygalacturonases. Npg1 is one of a two-member gene family expressed predominantly in the male gametophyte upon first microspore mitosis. From expression studies of promoter::GUS transgenes it is clear that the -744 bp to +74/+85 bp of Npg1 sequence (with respect to the transcription start site) is sufficient to drive the expression of the GUS reporter gene in a manner that reflects the spatial and temporal expression of Npg1 as determined by dot-blot and northern analysis.

Amino Acid Sequence↗
PubMed2002

Combinatorial interaction of cis elements specifies the expression of the Arabidopsis AtHsp90-1 gene.

The promoter region of the Arabidopsis AtHsp90-1 gene is congested with heat shock elements and stress response elements, as well as with other potential transcriptional binding sites (activating protein 1, CCAAT/enhancer-binding protein element, and metal regulatory element). To determine how the expression of this bona fide AtHsp90-1 gene is regulated, a comprehensive quantitative and qualitative promoter deletion analysis was conducted under various environmental conditions and during development. The promoter induces gene expression at high levels after heat shock and arsenite treatment. However, our results show that the two stress responses may involve common but not necessarily the same regulatory elements. Whereas for heat induction, heat shock elements and stress response elements act cooperatively to promote high levels of gene expression, arsenite induction seems to require the involvement of activating protein 1 regulatory sequences. In stressed transgenic plants harboring the full-length promoter, beta-glucuronidase activity was prominent in all tissues. Nevertheless, progressive deletion of the promoter decreases the level of expression under heat shock and restricts it predominantly in the two meristems of the plant. In contrast, under arsenite induction, proximal sequences induce AtHsp90-1 gene expression only in the shoot meristem. Distally located elements negatively regulate AtHsp90-1 gene expression under unstressed conditions, whereas flower-specific regulated expression in mature pollen grains suggests the prominent role of the AtHsp90-1 in pollen development. The results show that the regulation of developmental expression, suppression, or stress induction is mainly due to combinatorial contribution of the cis elements in the promoter region of the AtHsp90-1 gene.

Acclimatization↗