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Epitope analysis of birch pollen allergen in Japanese subjects.

Birch pollen is a very common cause of nasal allergy (pollinosis) not only in Scandinavia, Europe, Canada, and the northern part of the United States but also in Hokkaido, Japan. We have previously reported a positive association between the HLA-DR9 phenotype and the development of birch pollen allergy in Japanese subjects. However, there is little information about T cell epitopes of birch pollen which are presented by HLA class II molecules other than HLA-DR9. Therefore, we analyzed the difference in T cell epitope usage in patients who had HLA-DR9 versus those who did not. Seven Japanese patients with birch pollinosis were studied. Some groups of peptides representing T cell epitopes (Betula verrucosa; Bet VI peptides, p7-33, p23-46, p138-160) appeared to be shared by the majority, while another peptide (Bet VI p72-95) was recognized predominantly by patients who expressed HLA-DR9 and/or HLA-DQ3 molecules. Moreover, seven T cell clones and eight T cell lines were generated from two patients who did not have HLA-DR9 or HLA-DQ3. Using some of these T cell clones/lines, we investigated the relationship between HLA class II molecules and antigenic peptides. One of these T cell clones recognized antigenic peptides in the context of the HLA-DQ1 molecule. To our knowledge, this is the first indication that the epitope on Bet VI can be presented by the HLA-DQ molecule.

Allergens↗

[The pollen count of Cryptomeria japonica and nasal symptom of Cryptomeria japonica pollinosis patients in recent three years in Mie Prefecture].

Airborne scattering of Cryptomeria japonica pollen depends on climate. Total pollen count and scattering pattern varies from one year to another. We counted Cryptomeria japonica pollen in February and March from 1993 to 1995 at several points in Mie Prefecture. One-third to two-thirds of all the patients who visited our clinic for the first time in the pollen period were occupied by allergic patients. The pollen count in 1995 amounted to 17943/cm2 at Tsu and 99512/cm2 in the whole prefecture, which is the largest in the past 12 years. The number of days in which patients severely suffered was correlated to the pollen count for the past three years. It was shown that nasal symptoms developed after certain amount of pollen had scattered, but the severity of symptoms thereafter largely depended on the total pollen count. At Tsu in 1995, we scarcely had pollen scattering in February but had tremendous amount of pollen count in March. Percentage of development of nasal symptoms among thirty patients was well correlated to the logarithm of accumulated pollen count. When accumulated pollen count reached to 100/cm2, half the patients developed symptoms. Most of the patients developed symptoms when it reached to 1000/cm2. Since there is a great interindivisual difference in when and how much patients have nasal symptoms, the choice of therapy should be based not only on the pollen count but also on the symptom and condition of each patient.

Adult↗

A pollen-specific DEAD-box protein related to translation initiation factor eIF-4A from tobacco.

A pollen-specific sequence, NeIF-4A8, has been isolated from a cDNA library from mature pollen of Nicotiana tabacum cv. Samsun. NeIF-4A8 is a full-length cDNA whose deduced amino acid sequence exhibits high homology to the eucaryotic translation initiation factor eIF-4A from mouse, Drosophila and tobacco. eIF-4A is an RNA helicase which belongs to the supergene family of DEAD-box proteins. Northern blot analysis with a gene-specific probe showed strict anther-specific expression of NeIF-4A8 starting at microspore mitosis. With antibodies raised against tobacco eIF-4A the presence of abundant eIF-4A-related proteins in developing anthers and pollen grains was demonstrated. The genomic analysis shows that the coding region is split by three introns whereas a large, fourth intron is situated in the 5'-untranslated region. A promoter construct with 2137 bp of upstream sequence fused to the GUS reporter gene was used to confirm that the expression is confined to the haploid cells within the anther. NeIF-4A8 is a prime candidate formediating translational control in the developing male gametophyte.

Amino Acid Sequence↗

Use of novel pollen species by specialist and generalist solitary bees (Hymenoptera: Megachilidae).

If trade-offs between flexibility to use a range of host species and efficiency on a limited set underlie the evolution of diet breadth, one resulting prediction is that specialists ought to be more restricted than generalists in their ability to use novel resource species. I used foraging tests and feeding trials to compare the ability of a generalist and a specialist solitary mason bee species to collect and develop on two pollen species that are not normally used in natural populations (novel pollens). Osmia lignaria (Hymenoptera: Megachilidae) is a generalist pollen feeder; O. californica, is more specialized. Adults of the specialist were more limited in use of novel hosts, but only in some contexts. Both bee species refused to collect one novel pollen. The specialist accepted a second novel pollen only when it was presented along with its normal pollen, whereas the generalist collected novel pollen whether presented alone or with normal pollen. Surprisingly, larvae of the specialist were more flexible than were generalists. The specialist grew well on mixtures of normal and novel pollen species, in some cases better than on its normal host alone. Larvae of the generalist grew more poorly on all diets containing novel pollens than on their normal host. Data on these two species of bees suggest that specialization by itself need not reduce flexibility on novel hosts. The findings also provide information about mechanisms of specialization in bees. Similar to some folivores, specific cues of the pollen host and the bee's interpretation of these contribute, along with foraging economics, to pollen choice by adults. The ability of the larvae to cope with specific components of one pollen species need not interfere with its ability to use others.

Adaptation, Physiological↗

Ablation of Papillar Cell Function in Brassica Flowers Results in the Loss of Stigma Receptivity to Pollination.

Plant reproduction in crucifers is dependent on interactions that occur at the stigma surface between the male gametophyte (pollen and pollen tube) and papillar cells. To dissect these complex interactions, papillar cells were genetically ablated by targeting the expression of a toxin to appropriate cells of the flower with a flower-specific and developmentally regulated promoter. In transgenic Brassica plants that expressed the toxic gene fusion, flower morphology was normal except for aberrant papillar cell development and partial pollen sterility. Microscopic, biochemical, and functional analyses, mainly focused on papillar cell responses, revealed that papillar cells lost their ability to elongate, to synthesize cell-specific proteins, and to support pollen germination after self- or cross-pollination. This loss of stigma receptivity to pollination was mimicked by treating pistils with protein phosphatase inhibitors. Differences in the effects of genetic and chemical ablation on the pollination responses of Brassica and Arabidopsis flowers are discussed and are ascribed in part to a requirement for phosphorylation/dephosphorylation events in Brassica but not in Arabidopsis.

Journal Article↗

The genetic basis of naturally occurring pollen color dimorphisms in Nigella degenii (Ranunculaceae).

Nigella degenii ssp. barbro and ssp. jenny differ from related taxa in being dimorphic for pollen color, with some plants having dark pollen and others light pollen. In this study we performed experimental crosses to determine whether the difference in pollen color is governed by few or many loci and whether the two subspecies utilize the same gene to control pollen color. Patterns of segregation in crosses between morphs show that dark pollen is dominant over light pollen and that a single major gene is responsible for most of the variation in pollen color. Consequently it should be relatively easy for pollen color dimorphisms to establish and spread in these subspecies. Aberrant segregation ratios were attributed to genetic factors that reduced the expression of the allele conferring dark pollen or processes that sorted between color morphs during seed development. Crosses between dark pollen plants from different subspecies showed signs of complementation in the F2 generation, but the frequency of the light morph was too low to support a model involving complementary action of recessive alleles at two separate loci. Based on this and other observations, we hypothesize that the pollen color difference is controlled by the same major locus in the two subspecies.

Analysis of Variance↗

Analysis of natural history of Japanese cedar pollinosis.

BACKGROUND: The marked increase in the incidence of Japanese cedar (Cryptomeria japonica; JC) pollinosis is a social problem in Japan. Elucidation of its natural history is, therefore, essential. METHODS: Cross-sectional and vertical-sectional studies were performed regarding the effects of aging on sensitization to Japanese cedar pollen (JCP) and development of JC pollinosis by measuring serum IgE titers to JCP and by oral examination of residents of the Maruyama Town, Chiba, Japan from 1995 to 2001. We also studied the incidence of its spontaneous remission and the background factors. RESULTS: In a vertical-sectional study, the serum IgE titer to JCP was strongly influenced by the amounts of pollen scattered. An increase in age by 6 years did not reduce the IgE titer to JCP in subjects in their 40s. However, in subjects aged 60 or more, annual differences in the JCP count did not affect serum IgE titer to JCP, which remained low even after a season with a high pollen count. In subjects with JC pollinosis aged over 40 showing a CAP RAST score of more than 2 to JCP in 1995, spontaneous remission of JC pollinosis was observed in 16.1% over a period of 6 years. Factors affecting spontaneous remission include sex, age at the time, serum IgE titer to JCP and age at first onset of JC pollinosis. CONCLUSIONS: The CAP RAST score was strongly associated with spontaneous remission in the multivariable model.

Adolescent↗

Arabidopsis hapless mutations define essential gametophytic functions.

In flowering plants, the egg develops within a haploid embryo sac (female gametophyte) that is encased within the pistil. The haploid pollen grain (male gametophyte) extends a pollen tube that carries two sperm cells within its cytoplasm to the embryo sac. This feat requires rapid, precisely guided, and highly polarized growth through, between, and on the surface of the cells of the stigma, style, and ovary. Pollen tube migration depends on a series of long-range signals from diploid female cells as well as a short-range attractant emitted by the embryo sac that guides the final stage of tube growth. We developed a genetic screen in Arabidopsis thaliana that tags mutant pollen with a cell-autonomous marker carried on an insertion element. We found 32 haploid-disrupting (hapless) mutations that define genes required for pollen grain development, pollen tube growth in the stigma and style, or pollen tube growth and guidance in the ovary. We also identified genomic DNA flanking the insertion element for eleven hap mutants and showed that hap1 disrupts AtMago, a gene whose ortholog is important for Drosophila cell polarity.

Arabidopsis↗

Expression analyses of Arabidopsis oligopeptide transporters during seed germination, vegetative growth and reproduction.

AtOPT promoter-GUS fusions were constructed for six of the nine known, putative oligopeptide transporters (OPTs) in Arabidopsis thaliana and used to examine AtOPT expression at various stages of plant development. AtOPT1, AtOPT3, AtOPT4, AtOPT6 and AtOPT7 were expressed in the embryonic cotyledons prior to root radicle emergence. Except for AtOPT8, which gave weak expression, all AtOPTs were strongly expressed in post-germinative seedlings with strongest expression in vascular tissues of cotyledons and hypocotyls. Preferential expression of AtOPTs in vascular tissues was also observed in cotyledons, leaves, hypocotyls, roots, flowers, siliques, and seed funiculi of seedlings and adult plants. Differential tissue-specific expression was observed for specific AtOPTs. For example, AtOPT1, AtOPT3 and AtOPT8 were uniquely expressed in pollen. Only AtOPT1 was expressed in growing pollen tubes, while only AtOPT6 was observed in ovules. AtOPT8 was transiently expressed in seeds during early stages of embryogenesis. Iron limitation was found to enhance expression of AtOPT3. These data suggest distinct cellular roles for specific AtOPTs including nitrogen mobilization during germination and senescence, pollen tube growth, pollen and ovule development, seed formation and metal transport.

Arabidopsis↗

Four members of the maize beta-tubulin gene family are expressed in the male gametophyte.

Four different beta-tubulin cDNA clones have been identified in maize pollen cDNA libraries. Three of the four cDNA clones represent new maize beta-tubulin genes that have been designated tub3, tub4 and tub5. It is shown that the beta-tubulin gene family in maize is more complex than originally anticipated and more complex than those in vertebrates. In the maize beta-tubulin gene family the tub3, tub4 and tub5 genes are shown to represent small beta-tubulin gene subfamilies. Differences in the abundance of the tub3, tub4 and tub5 transcripts are seen among vegetative and reproductive tissues. The tub3 and tub4 transcripts are most abundant in pollen. In spikelet development, abundance of the tub3 transcript increases markedly from the 0.7 cm to the 1.0 cm spikelets whilst the levels of tub4 transcript closely resemble those of total beta-tubulin transcript in the organs examined. The tub3 and tub4 genes appear to have diverged recently in the evolution of the maize beta-tubulin gene family. The tub5 gene is expressed in pollen but its transcript is most abundant in maize vegetative tissues.

Amino Acid Sequence↗

A CDC45 homolog in Arabidopsis is essential for meiosis, as shown by RNA interference-induced gene silencing.

CDC45 is required for the initiation of DNA replication in yeast and cell proliferation in mammals and functions as a DNA polymerase alpha loading factor in Xenopus. We have cloned a CDC45 homolog from Arabidopsis whose expression is upregulated at the G1/S transition and in young meiotic flower buds. One-third of Arabidopsis 35S:CDC45 T1 RNA interference lines are partially to completely sterile, and the proportion of sterile plants is increased by using a dmc1 promoter. T1 plants have decreased levels of the CDC45 transcript and contain 21- to 23-bp RNA fragments specific to the CDC45 gene. T2 transgenic lines, in which small RNA fragments are still present, were used to analyze S-phase entry by 5-bromodeoxyuridine incorporation, which was not altered compared with that in the wild type. However, microarray data show that other cell cycle genes are upregulated or downregulated. T2 plants also have highly reduced fertility. The severity of the phenotype is correlated with the levels of the CDC45 transcript and small RNA fragments. Severe chromosome fragmentation arising during meiosis, which is not the result of a defect in the repair of SPO11-induced double strand breaks, leads to abnormal chromosome segregation and defective pollen and ovule development.

Arabidopsis↗

Enhanced development in nature of larval Anopheles arabiensis mosquitoes feeding on maize pollen.

To determine whether pollen produced by maize (Zea m. mays) may contribute to the development of larval Anopheles gambiae complex mosquitoes, the main African vectors of malaria, we correlated duration of larval development, pupation success, and size of the resulting adults with degree of access to this potential nutriment. Maize pollen is abundant during the wet season on the surface of water near maize plantings in a malaria-endemic region of Ethiopia, and larval Anopheles arabiensis readily ingest these particles in nature. Larvae develop to the pupal stage more rapidly, more frequently, and produce larger adults where maize pollen is abundant than do those that have little access to this food. The force of transmission of malaria in sub-Saharan Africa might be reduced if maize plantings were excluded from the immediate vicinity of homes or, perhaps, if pollen of such maize were to express entomotoxins.

Animals↗

Reduction in allergenicity of grass pollen by genetic engineering.

BACKGROUND: Hay fever and allergic asthma triggered by grass pollen allergens affect approximately 20% of the population in cool temperate climates. Ryegrass is the dominant source of allergens due to its prodigious airborne pollen production. Lol p 5 or group 5 is among the most important and widespread grass pollen allergen because it reacts with IgE antibodies of more than 90% of grass pollen-allergic patients, contains most of the grass pollen-specific IgE epitopes and elicits strong biological responses. Significant efforts have been made in developing diagnostic and therapeutic reagents for designing new and more effective immunotherapeutic strategies for treatment of allergic diseases. An alternative approach to this problem could be to reduce the amount of allergen content in the source plant. METHODS: High velocity microprojectile bombardment was used to genetically engineer ryegrass. Antisense construct targeted to one of major allergen, Lol p 5, was introduced. The expression of antisense RNA was regulated by a pollen-specific promoter. Pollen was analysed for IgE reactivity. RESULTS: Analysis of proteins with allergen-specific monoclonal and polyclonal 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 to control pollen. The transgenic ryegrass plants in which Lol p 5 gene expression is perturbed showed normal fertile pollen development. CONCLUSIONS: Our studies showed that it is possible to selectively 'switch off' allergen production in pollen of ryegrass demonstrating feasibility of genetic engineering of plants for reduced allergenicity.

Allergens↗

Cell-specific regulation of a Brassica napus CMS-associated gene by a nuclear restorer with related effects on a floral homeotic gene promoter.

Cytoplasmic male sterility (CMS) is a maternally inherited defect in pollen production specified by novel mitochondrial genes. It can be suppressed by nuclear restorer (Rf) genes which normally downregulate expression of a CMS-associated novel mitochondrial gene. Two forms of Brassica napus CMS, nap and pol, are associated with related chimeric genes, orf222 and orf224, respectively. We show that in pol and nap CMS, anther locule development is asynchronous and asymmetric, that one or more locules within each anther may fail to develop entirely and that CMS anthers display polarity in locule development. We show, by in situ hybridization, that orf222 transcripts accumulate in sterile anthers prior to development of morphological differences between CMS and restored stamens, and remain preferentially localized to microsporangia. In fertility-restored anthers, however, orf222 transcript levels remain low throughout development. Some sporogenous and meiotic cells differentiate within CMS anthers and form functional pollen despite retaining high orf222 transcript levels, suggesting that the effect of orf222 expression in blocking pollen development is limited to an early and specific stage. Transcripts of other mitochondrial genes, exemplified by atp6 and cob, and of the nuclear-encoded ATP synthase gamma subunit, accumulate preferentially in the microsporangia of both sterile and fertile anthers. Thus nuclear fertility restoration reduces orf222 transcript levels in a gene and tissue-specific manner. We observe differences between CMS and fertile plants in the timing and patterning of APETALA3 promoter activity that suggest a possible basis for the developmental abnormalities of CMS flowers.

Brassica napus↗

Development of a proxy for past surface UV-B irradiation: a thermally assisted hydrolysis and methylation py-GC/MS method for the analysis of pollen and spores.

A method was developed for the analysis of the UV-absorbing sporopollenin monomers p-coumaric acid and ferulic acid in very low numbers of pollen. This enables the analysis of pollen or spores from cultured plants, from herbarium collections, and from sediment, soil, and peat cores. The method involves thermally assisted hydrolysis and methylation using tetramethylammonium hydroxide combined with gas chromatography and mass spectrometry. Pyrolysis, gas chromatographic, and mass spectrometric conditions were optimized for the analysis of minimal amounts of pollen. The method has a detection limit of approximately 60 fresh pollen of Alnus glutinosa and a relative standard deviation of approximately 10% between 100 and 600 pollen.

Alnus↗

Peptide immunotherapy for allergic diseases using a rice-based edible vaccine.

PURPOSE OF REVIEW: Plant pollens are the most common cause of seasonal allergic disease. The number of patients undergoing treatment for allergies to the pollen of Japanese cedar (major antigens: Cry j 1 and Cry j 2) has increased steadily each year. Integration of an effective, safe and inexpensive clinical program would be greatly improved by addressing deficiencies in systemically delivered immunotherapy. RECENT FINDINGS: We have demonstrated that feeding mice transgenic rice seeds accumulating the T-cell epitope peptides of Cry j 1 and Cry j 2 before systemic challenge with total protein of cedar pollen inhibits the development of allergen-specific IgE, IgG and CD4 T-cell proliferative responses. The levels of allergen-specific CD4 T-cell-derived allergy-associated T-helper 2 cytokine of IL-4, IL-5, and IL-13 and histamine release in serum were also significantly decreased. Moreover, clinical symptoms were inhibited in an experimental sneezing-mouse model. SUMMARY: Plant-based edible vaccine has been shown to be effective for treatment of Japanese cedar pollinosis. When rice seeds containing T-cell epitopes derived from cedar pollen allergens were orally administered to mice, immune tolerance leading to reduction of allergen-specific IgE, T-cell proliferative reaction and histamine could be induced, resulting in suppression of allergic-specific symptoms such as sneezing.

Administration, Oral↗

Pollen tubes of Nicotiana alata express two genes from different beta-glucan synthase families.

The walls deposited by growing pollen tubes contain two types of beta-glucan, the (1,3)-beta-glucan callose and the (1,4)-beta-glucan cellulose, as well as various alpha-linked pectic polysaccharides. Pollen tubes of Nicotiana alata Link et Otto, an ornamental tobacco, were therefore used to identify genes potentially encoding catalytic subunits of the callose synthase and cellulose synthase enzymes. Reverse transcriptase-polymerase chain reactions (RT-PCR) with pollen-tube RNA and primers designed to conserved regions of bacterial and plant cellulose synthase (CesA) genes amplified a fragment that corresponded to an abundantly expressed cellulose-synthase-like gene named NaCslD1. A fragment from a true CesA gene (NaCesA1) was also amplified, but corresponding cDNAs could not be identified in a pollen-tube library, consistent with the very low level of expression of the NaCesA1 gene. RT-PCR with pollen-tube RNA and primers designed to regions conserved between the fungal FKS genes [that encode (1,3)-beta-glucan synthases] and their presumed plant homologs (the Gsl or glucan-synthase-like genes) amplified a fragment that corresponded to an abundantly expressed gene named NaGsl1. A second Gsl gene detected by RT-PCR (NaGsl2) was expressed at low levels in immature floral organs. The structure of full-length cDNAs of NaCslD1, NaCesA1, and NaGsl1 are presented. Both NaCslD1 and NaGsl1 are predominantly expressed in the male gametophyte (developing and mature pollen and growing pollen tubes), and we propose that they encode the catalytic subunits of two beta-glucan synthases involved in pollen-tube wall synthesis. Different beta-glucans deposited in one cell type may therefore be synthesized by enzymes from different gene families.

Amino Acid Sequence↗

CaMADS1, a MADS box gene expressed in the carpel of hazelnut.

Hazelnut (Corylus avellana L.) is a species of economic interest that shows a peculiar floral biology. Unlike most of the angiosperms, which produce ovules during floral development such that they are ready for pollen at anthesis, hazelnut ovary development is delayed and triggered by compatible pollination. In order to elucidate the mechanisms regulating this unusual process and the role of the MADS box genes in ovary development, a cDNA library from pollinated styles of hazelnut was screened with a mixture of MADS box genes from different plant species. CaMADS1 (Corylus avellana MADS box), a floral-specific MADS box gene, was isolated, and characterized as belonging to the sub-family of the AGAMOUS genes. Northern blot, RT-PCR analyses and in situ hybridization experiments show a precise correlation between ovary development and CaMADS1 expression, indicating a role of this MADS box gene in the processes of floral organogenesis.

Amino Acid Sequence↗