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PubMed2003

AtSTP6, a new pollen-specific H+-monosaccharide symporter from Arabidopsis.

This paper describes the molecular, kinetic, and physiological characterization of AtSTP6, a new member of the Arabidopsis H(+)/monosaccharide transporter family. The AtSTP6 gene (At3g05960) is interrupted by two introns and encodes a protein of 507 amino acids containing 12 putative transmembrane helices. Expression in yeast (Saccharomyces cerevisiae) shows that AtSTP6 is a high-affinity (K(m) = 20 microM), broad-spectrum, and uncoupler-sensitive monosaccharide transporter that is targeted to the plasma membrane and that can complement a growth deficiency resulting from the disruption of most yeast hexose transporter genes. Analyses of AtSTP6-promoter::GUS plants and in situ hybridization experiments detected AtSTP6 expression only during the late stages of pollen development. A transposon-tagged Arabidopsis mutant was isolated and homozygous plants were analyzed for potential effects of the Atstp6 mutation on pollen viability, pollen germination, fertilization, and seed production. However, differences between wild-type and mutant plants could not be observed.

Arabidopsis↗

PubMed2003

In silico identification of putative regulatory sequence elements in the 5'-untranslated region of genes that are expressed during male gametogenesis.

During pollen development, transcription of a large number of genes results in the appearance of distinct sets of transcripts. Similar mRNA sets are present in pollen of both mono- and dicotyledonous plant species, which indicates an evolutionary conservation of genetic programs that determine pollen gene expression. In pollen, regulation of gene expression occurs at the transcriptional and posttranscriptional level. The 5'-untranslated region (UTR) of several pollen transcripts has been shown to be important for regulation of pollen gene expression. The important regulatory role of 5'-UTR sequences and the evolutionary conservation of genetic programs in pollen led to the hypothesis that the 5'-UTRs of pollen-expressed genes share regulatory sequence elements. In an attempt to identify these pollen 5'-UTR elements, a statistical analysis was performed using 5'-UTR sequences of pollen- and sporophytic-expressed genes. The analysis revealed the presence of several pollen-specific 5'-UTR sequence elements. Assembly of the pollen 5'-UTR elements led to the identification of various consensus sequences, including those that previously have been demonstrated to play a role in the regulation of pollen gene expression. Several pollen 5'-UTR elements were found to be preferentially associated to genes from dicots, wet-type stigma plants, or plants containing bicellular pollen. Moreover, three sequence elements exhibited a preferential association to the 5'-UTR of pollen-expressed genes from Arabidopsis and Brassica napus. Functional implications of these observations are discussed.

5' Untranslated Regions↗

PubMed2003

The tomato homolog of CORONATINE-INSENSITIVE1 is required for the maternal control of seed maturation, jasmonate-signaled defense responses, and glandular trichome development.

Jasmonic acid (JA) is a fatty acid-derived signaling molecule that regulates a broad range of plant defense responses against herbivores and some microbial pathogens. Molecular genetic studies in Arabidopsis have established that JA also performs a critical role in anther and pollen development but is not essential for other developmental aspects of the plant's life cycle. Here, we describe the phenotypic and molecular characterization of a sterile mutant of tomato (jasmonic acid-insensitive1 [jai1]) that is defective in JA signaling. Although the mutant exhibited reduced pollen viability, sterility was caused by a defect in the maternal control of seed maturation, which was associated with the loss of accumulation of JA-regulated proteinase inhibitor proteins in reproductive tissues. jai1 plants exhibited several defense-related phenotypes, including the inability to express JA-responsive genes, severely compromised resistance to two-spotted spider mites, and abnormal development of glandular trichomes. We demonstrate that these defects are caused by the loss of function of the tomato homolog of CORONATINE-INSENSITIVE1 (COI1), an F-box protein that is required for JA-signaled processes in Arabidopsis. These findings indicate that the JA/COI1 signaling pathway regulates distinct developmental processes in different plants and suggest a role for JA in the promotion of glandular trichome-based defenses.

Amino Acid Sequence↗

PubMed1996

A 610 kb YAC clone harbors 7 cM of tomato (Lycopersicon esculentum) DNA that includes the male sterile 14 gene and a hotspot for recombination.

Pollen development requires both sporophytic and gametophytic gene expression. We are using a map-based cloning technique to isolate sporophytic genes which, when mutant, cause pollen abortion and a male sterile (ms) phenotype in tomato (Lycopersicon esculentum). We have genetically characterized one ms locus (ms14) using RFLP analysis and identified flanking markers. High-resolution genomic physical mapping indicates that the ms14 locus is located in a approximately 300 kb region. We have identified a YAC clone with an insert size of approximately 610 kb that contains the ms14-linked markers, reflects the organization of the physical map and therefore most probably contains the ms14 gene. In addition, we present evidence that the relationship between physical and genetic distance in this chromosomal region changes abruptly from approximately 105-140 kb/cM to less than 24kb/cM, and suggest that the TG393-TG104 region is a hotspot for recombination.

Chromosome Mapping↗

PubMed1999

The late pollen-specific actins in angiosperms.

The actin gene family of Arabidopsis has eight functional genes that are grouped into two ancient classes, vegetative and reproductive, and into five subclasses based on their phylogeny and mRNA expression patterns. Progress in deciphering the functional significance of this diversity is hindered by the lack of tools that can distinguish the highly conserved subclasses of actin proteins at the biochemical and cellular level. In order to address the functional diversity of actin isovariants, we have used Arabidopsis recombinant actins as immunogens and produced several new anti-actin monoclonal antibodies. One of them, MAb45a, specifically recognizes two closely related reproductive subclasses of actins. On immunoblots, MAb45a reacts strongly with actins expressed in mature pollen but not with actins in other Arabidopsis tissues. Moreover, immunocytochemical studies show that this antibody can distinguish actin filaments in pollen tubes from those in most vegetative tissues. Peptide competition analyses demonstrate that asparagine at position 79 (Asn79) within an otherwise conserved sequence is essential for MAb45a specificity. Actins with the Asn79 epitope are also expressed in the mature pollen from diverse angiosperms and Ephedra but not from lower gymnosperms, suggesting that this epitope arose in an ancestor common to angiosperms and advanced gymnosperms more than 220 million years ago. During late pollen development in angio- sperms there is a switch in expression of actins from vegetative to predominantly reproductive subclasses, perhaps to fulfil the unique functions of pollen in fertilization.

Actins↗

PubMed1999

Ultrastructural distribution of a MAP kinase and transcripts in quiescent and cycling plant cells and pollen grains.

Mitogen-activated protein kinases (MAPKs) are components of a kinase module that plays a central role in the transduction of diverse extracellular stimuli, including mitogens, specific differentiation and developmental signals and stress treatments. This shows that reversible protein phosphorylation cascades play a pivotal role in signal transduction in animal cells and yeast, particularly the entry into mitosis of arrested cells. Homologues of MAPKs have been found and cloned in various plant species, but there have been no data about their in situ localization at the subcellular level and their expression in plant cells so far. In the present paper we report the first data on the ultrastructural in situ localization of MAPK and their mRNAs in various plant cells. Proliferating and quiescent meristematic plant cells were studied to evaluate whether changes in MAPK presence, distribution and expression accompany the entry into proliferation of dormant cells. Moreover, MAPK localization was analyzed in vacuolate microspores. Polyclonal antibodies against the deduced MAPK from the tobacco Ntf6 clone were able to recognize homologue epitopes by immunocytochemical techniques in the cell types studied. The pattern of protein distribution is similar in all the cases studied: it is localized in the cytoplasm and in the nucleus, mainly in the interchromatin region. The quantitative study of the density showed that MAPK labelling is more abundant in cycling than in quiescent cells, also suggesting that, in plants, MAPK pathways might play a role in cell proliferation. RNA probes for conserved regions of the catalytic domain of plant MAPK homologue genes were used to study MAPK expression in those plant cells. In situ hybridization (ISH) showed the presence of MAPK transcripts in the three plant cell types studied, but levels were very low in quiescent cells compared to those in cycling cells. The quantification of labelling density of ISH signals strongly suggests a higher level of MAPK expression in proliferating cells, but also some basal messenger presence and/or expression in the quiescent ones. Immunogold and ISH results show the presence and distribution of MAPK proteins and mRNAs in vacuolate microspores. This represents a very dynamic stage during pollen development in which the cell nucleus is being prepared for an asymmetrical mitotic division, giving rise to both the generative and the vegetative nuclei of the bicellular pollen grain. Taken together, the data indicate a role played by MAPK in the re-entry into proliferation in plant cells.

Calcium-Calmodulin-Dependent Protein Kinases↗

PubMed2001

Novel anther-specific myb genes from tobacco as putative regulators of phenylalanine ammonia-lyase expression.

Two cDNA clones (NtmybAS1 and NtmybAS2) encoding MYB-related proteins with strong sequence similarity to petunia (Petunia hybrida) PhMYB3 were isolated from a tobacco (Nicotiana tabacum cv Samsun) pollen cDNA library. Northern blot and in situ hybridization revealed that NtmybAS transcripts are specifically expressed in both sporophytic and gametophytic tissues of the anther including tapetum, stomium, vascular tissue, and developing pollen. Random binding site selection assays revealed that NtMYBAS1 bound to DNA sequences closely resembling consensus MYB binding sites MBSI and MBSIIG, with a higher affinity for MBSI. Transient expression analyses of the N-terminal MYB domain demonstrated the presence of functional nuclear localization signals, and full-length NtMYBAS1 was able to activate two different phenylalanine ammonia-lyase promoters (PALA and gPAL1) in tobacco leaf protoplasts. Similar analysis of truncated NtmybAS1 cDNAs identified an essential, C-terminal trans-activation domain. Further in situ hybridization analyses demonstrated strict co-expression of NtmybAS and gPAL1 in the tapetum and stomium. Despite abundant expression of NtmybAS transcripts in mature pollen, gPAL1 transcripts were not detectable in pollen. Our data demonstrate that NtMYBAS1 is a functional anther-specific transcription factor, which is likely to be a positive regulator of gPAL1 expression and phenylpropanoid synthesis in sporophytic, but not in gametophytic, tissues of the anther.

Amino Acid Sequence↗

PubMed1993

Characterization of a new class of oleosins suggests a male gametophyte-specific lipid storage pathway.

The expression and sequences of two related antherspecific cDNAs (I3 and C98) of Brassica napus (oilseed rape) were examined. These cDNAs were found to exhibit significant predicted amino acid sequence similarity with a group of seed-specific proteins, the oleosins, which are involved in oil body membrane structure. Pollen of B. napus also contains oil bodies, and the synthesis and accumulation of these organelles correlates with expression of the I3 and C98 transcripts. The protein content of purified pollen oil bodies was therefore examined in order to determine the presence or absence of possible oleosins. One major protein species of 14 kDa was identified and subjected to N-terminal amino acid sequence determination. The sequence of this pollen oil body associated protein is homologous to the predicted sequence of one of the anther-specific cDNs, I3, implying that this cDNA represents an anther-specific oleosin gene expressed in developing pollen. The expression of other genes active during seed oil body formation was also examined, and these were found to be specific to the sporophyte, inferring the existence of specific components of the pathway of lipid synthesis and storage in the male gametophyte. These possibilities and the relationship between different oleosin genes are discussed.

Amino Acid Sequence↗

PubMed2001

Pollen-specific regulation of vacuolar H+-PPase expression by multiple cis-acting elements.

We dissected the regulatory region of the AVP1 gene encoding the vacuolar H+-pyrophosphatase (V-PPase) of Arabidopsis thaliana by using a GUS-reporter assay system. The cloned 1.4 kb 5'-regulatory region in the GUS-reporter transgenic plants was sufficient for the light-induced repression. Furthermore, the 1.4 kb regulatory region was active in all tissues examined and its activity was especially enhanced in pollen, whereas the shorter 0.4 kb regulatory region was active only in pollen. Further detailed analyses revealed that the GUS activity in pollen was regulated by at least three cis-acting regions in an additive or synergetic manner. These findings establish a distinct mechanism of the tissue-specific regulation of V-PPase expression in developing pollen. and imply the biological significance of the V-PPase in pollen maturation.

Arabidopsis↗

PubMed1995

Characterization and expression of rpoC2 in CMS and fertile lines of sorghum.

A 165 bp deletion in the middle of rpoC2, the plastid gene which encodes the RNA polymerase beta" subunit, was identified in the small-anthered types of CMS sorghum, Sorghum bicolor (L.). Moench, containing A1, A2, A5, and A6 cytoplasms. It was previously shown that the amino acid sequence deleted in these CMS lines is in a monocot-specific region that contains several protein motifs that are characteristic of several transcription factors. Using primers flanking the deletion in PCR analyses, various types of CMS lines, some of which are used in hybrid sorghum production, were classified into two groups. CMS lines containing A1, A2, A5, A6 cytoplasms display the deletion in rpoC2. These lines have small anthers in which pollen development is arrested at an early stage and in which usually only empty exines are found. CMS lines containing A3, A4, and 9E cytoplasms do not possess the deletion. These lines have large anthers in which pollen degenerates at a later stage. Run-on transcription assays using 15 chloroplast genes showed that chloroplast gene transcription rates are similar in CMS and fertile (maintainer and restorer) lines and F1 in seedling leaves. Analyses of RNA blots indicated that rbcL, rpoB and rpoC2 transcripts are accumulated mainly in the leaves and low in the inflorescence tissues and pollen. These data document plastid gene expression in leaves and non-photosynthetic tissues from CMS and fertile lines of sorghum.

Base Sequence↗

PubMed1994

Sensitivity to olea Europaea pollen in different populations in Israel.

Sensitivity to olive pollen was studied (by skin tests) in different Israeli populations suffering from respiratory allergies. The results were compared with aerobiological data, in order to correlate amount of exposure to prevalence of sensitization. It was found that in the Jewish population, sensitivity to olive pollen developed in direct proportion with the number of olive trees in the settlement: 66% where trees density is high, 29% where it is low. In the Arab population however, possibly genetically determined, the sensitization to olive pollen was low (only 14%) even though the density of trees is high.

Allergens↗

PubMed2007

In situ molecular identification of the Ntf4 MAPK expression sites in maturing and germinating pollen.

BACKGROUND INFORMATION: MAPKs (mitogen-activated protein kinases) are involved in the transduction of different signals in eukaryotes. They regulate different processes, such as differentiation, proliferation and stress response. MAPKs act through the phosphorylation cascade, being the last element that phosphorylates the final effector of the cell response. They are activated when their threonine and tyrosine residues are phosphorylated. Ntf4, a MAPK with a molecular mass of 45 kDa, has been reported to be expressed in pollen and seeds. Biochemical studies have indicated that the expression and the activation of Ntf4 is regulated during pollen maturation, although an increase of the activation is observed when the pollen is hydrated, just at the beginning of the germination. However, nothing is known about its subcellular localization. RESULTS: In the present study, the in situ expression and subcellular localization of Ntf4 have been analysed during the tobacco pollen developmental pathway. Cryosections, freeze-substitution and cryo-embedding in Lowicryl K4M were used as processing techniques for subsequent immunofluorescence, immunogold labelling and in situ hybridization assays. During pollen maturation, Ntf4 showed an increase in expression, as demonstrated by in situ hybridization, and specific subcellular distributions. We found that the protein was expressed from mid bicellular pollen stage until the pollen was mature. In germinating pollen, the protein increased after the initiation of germination. Translocation of the protein to the nucleus was found at specific stages; the presence of Ntf4 in the nucleus was found in the last stage of the pollen maturation and in germinating pollen. Double immunofluorescence and immunogold labelling with anti-Ntf4 (AbC4) and anti-P-MAPK (phosphorylated MAPK) antibodies revealed the co-localization of both epitopes in the nucleus at late developmental stages. CONCLUSIONS: The temporal and spatial pattern of the expression sites of Ntf4 has been characterized during pollen development, indicating that Ntf4 is a 'late gene' that is upregulated during maturation and germination, with a possible role in the gametophytic function. The translocation of the Ntf4 protein from the cytoplasm to the nucleus at late pollen developmental stages, and its co-localization with the P-MAPK epitope in several nuclear sites, indicates a relationship between the Ntf4 nuclear translocation and its active state.

Antibodies↗

PubMed1987

Treatment of hay fever with sodium cromoglycate, hyposensitization, or a combination.

In 77 consecutive patients with grass pollen rhinitis as the only allergic disease, treatment with sodium cromoglycate (SCG) nasal spray, hyposensitization with aluminium hydroxide adsorbed extract of grass pollen as perennial treatment, and a combination of the two was compared. Specific serum-IgE, IgG and IgG4 were determined in patients who participated in three consecutive seasons. Significant immune response was found in the hyposensitization group, but not in the group treated with SCG. No correlation between the immunoglobulin responses and the individual symptom and/or medicine scores was found. Symptom and medicine scores were analysed in the 3 weeks with the highest pollen counts each year during 4 consecutive years. Higher scores were found in the group treated with sodium cromoglycate compared with the other two groups, but the difference was only significant for eye and total symptoms in the first season. None of the groups, however, suffered from more than mild to moderate symptoms even in the weeks with peak pollen counts. A significantly larger decrease in the wheals of the skin prick test was shown during hyposensitization than during treatment with SCG. No difference was found in the frequency of patients who became symptom-free or developed pollen asthma.

Clinical Trials as Topic↗

PubMed1999

Antisense-mediated silencing of a gene encoding a major ryegrass pollen allergen.

Type 1 allergic reactions, such as hay fever and allergic asthma, triggered by grass pollen allergens are a global health problem that affects approximately 20% of the population in cool, temperate climates. Ryegrass is the dominant source of allergens because of its prodigious production of airborne pollen. Lol p 5 is the major allergenic protein of ryegrass pollen, judging from the fact that almost all of the individuals allergic to grass pollen show presence of serum IgE antibodies against this protein. Moreover, nearly two-thirds of the IgE reactivity of ryegrass pollen has been attributed to this protein. Therefore, it can be expected that down-regulation of Lol p 5 production can significantly reduce the allergic potential of ryegrass pollen. Here, we report down-regulation of Lol p 5 with an antisense construct targeted to the Lol p 5 gene in ryegrass. The expression of antisense RNA was regulated by a pollen-specific promoter. Immunoblot analysis of proteins with allergen-specific antibodies did not detect Lol p 5 in the transgenic pollen. The transgenic pollen showed remarkably reduced allergenicity as reflected by low IgE-binding capacity of pollen extract as compared with that of control pollen. The transgenic ryegrass plants in which Lol p 5 gene expression is perturbed showed normal fertile pollen development, indicating that genetic engineering of hypoallergenic grass plants is possible.

Allergens↗

PubMed2006

Abolition of the tapetum suicide program ruins microsporogenesis.

Microsporogenesis in angiosperms takes places within the anther. Microspores are surrounded by a layer of cells, the tapetum, which degenerates during the later stages of pollen development with cytological features characteristic of programmed cell death (PCD). We report herein that the expression of AtBI-1, which suppresses Bax-induced cell death, in the tapetum at the tetrad stage inhibits tapetum degeneration and subsequently results in pollen abortion, while activation of AtBI-1 at the later stage does not. Our results demonstrate that the PCD signal commences at the tetrad stage and that the proper timing of PCD in the tapetum is essential for normal microsporogenesis.

Animals↗

PubMed2004

A model system to study the environment-dependent expression of the Bet v 1a gene encoding the major birch pollen allergen.

BACKGROUND: The major birch pollen allergen Bet v 1 (or Bet v 1a) is one of the main causes of seasonal type I allergies. Various environmental factors such as light, temperature and air pollution may influence the activity of the Bet v 1a gene. The creation of a model system to evaluate the role of environmental factors affecting the Bet v 1a gene expression would be highly desirable. We suggest the use of transgenic tobacco plants carrying a Bet v 1a promoter-reporter gene fusion as such a system. METHODS: The promoter of the Bet v 1a gene was isolated with the use of the Universal Genome Walker kit (BD Biosciences Clontech, USA). Web Software was used to search for putative cis-regulatory elements within the promoter. Transgenic tobacco plants harboring the promoter-beta-glucuronidase (GUS) reporter gene fusion were obtained via Agrobacterium tumefaciens-mediated transformation. Promoter activity was examined with histochemical and quantitative assays. RESULTS: Structural analysis predicted elements responsible for pollen-specific, light-, stress- and hormone-mediated induction within the Bet v 1a promoter. The evaluation of GUS activity in transgenic tobacco plants showed that the Bet v 1a promoter is pollen-specific. Moreover, the Bet v 1a promoter is considered to be the strongest isolated pollen-specific promoter reported to date. It was shown that temperature and abscisic acid positively regulate the activity of the Bet v 1a promoter during pollen development, providing evidence for environment-dependent regulation of the Bet v 1a gene. CONCLUSIONS: A model system to study the effect of environmental factors on the expression of the Bet v 1a gene encoding the major birch allergen in pollen was generated. Additionally, we suggest that this system could be used to search for factors that inhibit the activity of the gene in pollen in order to reduce the potential allergenicity of birch trees.

Abscisic Acid↗

PubMed2007

BcMF11, a putative pollen-specific non-coding RNA from Brassica campestris ssp. chinensis.

A full-length cDNA, BcMF11, has been cloned from Chinese cabbage (Brassica campestris L. ssp. chinensis Makino) using rapid amplification of the cDNA ends (RACE) based on a pollen-specific cDNA fragment (DN237921). The BcMF11 cDNA has a total length of 828bp with poly (A) tail. Analysis of the sequence demonstrated that BcMF11 is a novel non-coding RNA which has no prominent open reading frame (ORF) or coding capacity. No significant similarities were observed between BcMF11 and previously published sequences in GenBank. Transcription analysis indicated that BcMF11 is a novel pollen-specific ncRNA and may be involved in the pollen development of Chinese cabbage.

Base Sequence↗

PubMed2005

Plant separation: 50 ways to leave your mother.

One of the remarkable features of plants is their ability to shed organs, such as leaves, seeds, flowers, and fruit. Genetic analysis of fruit dehiscence and floral organ shedding in Arabidopsis is revealing the pathways that underlie these distinct separation events. The transcriptional network that patterns the fruit links factors that regulate organ polarity and growth with those that control differentiation of the three cell types that are required for dehiscence. Transcriptional regulators that pattern the proximal-distal axis in developing leaves are required for floral organ shedding, and chromatin-modifying complexes might globally regulate genes that affect flower senescence and abscission. Ground-breaking studies have also recently identified a hydrolytic enzyme that is required for microspore separation during pollen development, and the first transcription factor controlling seed abscission.

Flowers↗