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PubMed1997

Maturation and secretion of a serine proteinase is associated with events of late microsporogenesis.

An antiserum against meiotic proteins which bind to DNA cellulose was generated as a tool to assist the identification and purification of microsporogenesis-specific proteins. In immunoblotting experiments, this antiserum identified three meiotic proteins which are differentially expressed in anthers during microsporogenesis. One of these proteins was purified and characterized by biochemical and immunological techniques. This 82 kDa protein is synthesized as a preproprotein, acquires glycans as it moves through the endoplasmic reticulum and Golgi body, and is secreted into the anther locule. Immunocytochemical experiments demonstrate that the protein is expressed primarily in tapetal cells, and reaches peak concentrations as the microsporocytes reach the tetrad stage. Zymogram analyses and protein sequence comparisons indicate that the protein is a member of the serine proteinase family. The possible roles of the proteinase in microsporogenesis and pollen development are discussed.

Amino Acid Sequence↗
PubMed2002

A knock-out mutation in allene oxide synthase results in male sterility and defective wound signal transduction in Arabidopsis due to a block in jasmonic acid biosynthesis.

Recent studies on jasmonic acid (JA) biosynthetic mutants have shown that jasmonates play essential roles in pollen maturation and dehiscence and wound-induced defence against biotic attacks. To better understand the biosynthetic mechanisms of this essential plant hormone, we isolated an Arabidopsis knock-out mutant defective in the JA biosynthetic gene CYP74A (allene oxide synthase, AOS) using reverse genetics screening methods. This enzyme catalyses dehydration of the hydroperoxide to an unstable allene oxide in the JA biosynthetic pathway. Endogenous JA levels, which increase 100-fold 1 h after wounding in wild-type plants, do not increase after wounding in the aos mutant. In addition, the mutant showed severe male sterility due to defects in anther and pollen development. The male-sterile phenotype was completely rescued by exogenous application of methyl jasomonate and by complementation with constitutive expression of the AOS gene. RT-PCR analysis showed that the induction of transcripts for vegetative storage protein and lipoxygenase genes, previously shown to be inducible by wound and jasmonate application in the wild-type, was absent in the aos mutant. In transgenic plants constitutively expressing AOS, wound-induced JA levels were 50-100% higher compared to wild-type plants. Taken together with JA deficiency in the aos mutant, our results show that AOS is critical for the biosynthesis of all biologically active jasmonates. Our results also suggest that AOS expression is limiting JA levels in wounded plants, but that the AOS hydroperoxide substrate levels, controlled by upstream enzymes (lipoxygenase and phospholipase), determine JA levels in unwounded plants.

Arabidopsis↗
PubMed2002

COI1 links jasmonate signalling and fertility to the SCF ubiquitin-ligase complex in Arabidopsis.

Jasmonates (JAs) regulate Arabidopsis thaliana wound and defence responses, pollen development, and stress-related growth inhibition. Significantly, each of these responses requires COI1, an F-box protein. Other F-box proteins interact with SKP1 and cullin proteins to form SCF complexes that selectively recruit regulatory proteins targeted for ubiquitination. To determine whether COI1 also functions in an SCF complex, we have characterized Arabidopsis proteins that bind to COI1. An Arabidopsis cDNA expression library was screened in yeast for clones that produce proteins which can bind to COI1. We recovered two SKP1 homologues and a histone deacetylase. The Arabidopsis F-box protein TIR1 interacted with SKP1 proteins, but not with the histone deacetylase. Mutant COI1 proteins revealed that the F-box is required for interaction with SKP1s, but that sequences in leucine-rich repeat domains are required for interaction with the histone deacetylase. Epitope-tagged COI1 was introduced into Arabidopsis plants and cell cultures. Co-immunoprecipitation experiments confirmed the interaction in planta of COI1 with SKP1-like proteins and histone deacetylase, and also indicated that COI1 interacted with cullin. These results suggest that COI1 forms an SCFCOI1 complex in vivo. COI1 is therefore expected to form a functional E3-type ubiquitin ligase in plants and to regulate expression of jasmonate responsive genes, possibly by targeted ubiquitination of a histone deacetylase.

Amino Acid Sequence↗
PubMed2003

Carbon metabolite sensing and signalling.

The regulation of carbon metabolism in plant cells responds sensitively to the levels of carbon metabolites that are available. The sensing and signalling systems that are involved in this process form a complex web that comprises metabolites, transporters, enzymes, transcription factors and hormones. Exactly which metabolites are sensed is not yet known, but candidates include sucrose, glucose and other hexoses, glucose-6-phosphate, trehalose-6-phosphate, trehalose and adenosine monophosphate. Important components of the signalling pathways include sucrose non-fermenting-1-related protein kinase-1 (SnRK1) and hexokinase; sugar transporters are also implicated. A battery of genes and enzymes involved in carbohydrate metabolism, secondary metabolism, nitrogen assimilation and photosynthesis are under the control of these pathways and fundamental developmental processes such as germination, sprouting, pollen development and senescence are affected by them. Here we review the current knowledge of carbon metabolite sensing and signalling in plants, drawing comparisons with homologous and analogous systems in animals and fungi. We also review the evidence for cross-talk between carbon metabolite and other major signalling systems in plant cells and the prospects for manipulating this fundamentally important aspect of metabolic regulation for crop improvement.

Journal Article↗
PubMed2002

A pentatricopeptide repeat-containing gene restores fertility to cytoplasmic male-sterile plants.

Known in over 150 species, cytoplasmic male sterility is encoded by aberrant mitochondrial genes that prevent pollen development. The RNA- or protein-level expression of most of the mitochondrial genes encoding cytoplasmic male sterility is altered in the presence of one or more nuclear genes called restorers of fertility that suppress the male-sterile phenotype. Cytoplasmic male sterility/restorer systems have been proven to be an invaluable tool in the production of hybrid seeds. Despite their importance for both the production of major crops such as rice and sunflower and the study of organelle/nuclear interactions in plants, none of the nuclear fertility-restorer genes that reduce the expression of aberrant mitochondrial proteins have previously been cloned. Here we report the isolation of a gene directly involved in the control of the expression of a cytoplasmic male sterility-encoding gene. The Petunia restorer of fertility gene product is a mitochondrially targeted protein that is almost entirely composed of 14 repeats of the 35-aa pentatricopeptide repeat motif. In a nonrestoring genotype we identified a homologous gene that exhibits a deletion in the promoter region and is expressed in roots but not in floral buds.

Amino Acid Sequence↗
PubMed2003

Fungicide sprays can injure the stigmatic surface during receptivity in almond flowers.

Fungicides can be detrimental to flower development, pollen function and fruit set in a number of crops. Almond is a self-incompatible nut crop that has a fruit set of only approx. 30 % of the total number of flowers. Thus, interference of pollination and fertilization by fungicide sprays is of concern, and identification of chemicals having the least detrimental effects would be desirable. The objective of this study was to evaluate the effect of fungicide sprays on stigma morphology in almond using a laboratory spray apparatus that simulated field applications. Four fungicides (azoxystrobin, myclobutanil, iprodione and cyprodinil) were applied, and fresh, unfixed stigmatic surfaces were observed using a scanning electron microscope at 4 and 24 h after spraying. Increased exudate accumulation was induced by azoxystrobin at both time periods, and localized damage and collapse of stigmatic cells were observed after 24 h. Damaged stigmatic papillae exhibited wrinkling, surface distortion or collapse. Likewise, myclobutanil caused significant damage to and collapse of papillae; these were more extensive at later observations. Iprodione had no effect on exudate accumulation but caused marked and severe collapse of stigmatic papillae which was pronounced at 24 h. Cyprodinil promoted a copious increase in exudate secretion and caused the most severe collapse of stigmatic cells of all the fungicides evaluated. Damage was somewhat localized at 4 h but more global at 24 h. This study has verified that certain fungicide sprays have direct detrimental effects on stigma morphology and enhance exudate production in almond flowers.

Flowers↗
PubMed2001

Localization of male-specifically expressed MROS genes of Silene latifolia by PCR on flow-sorted sex chromosomes and autosomes.

The dioecious white campion Silene latifolia (syn. Melandrium album) has heteromorphic sex chromosomes, XX in females and XY in males, that are larger than the autosomes and enable their separation by flow sorting. The group of MROS genes, the first male-specifically expressed genes in dioecious plants, was recently identified in S. latifolia. To localize the MROS genes, we used the flow-sorted X chromosomes and autosomes as a template for PCR with internal primers. Our results indicate that the MROS3 gene is located in at least two copies tandemly arranged on the X chromosome with additional copy(ies) on the autosome(s), while MROS1, MROS2, and MROS4 are exclusively autosomal. The specificity of PCR products was checked by digestion with a restriction enzyme or reamplification using nested primers. Homology search of databases has shown the presence of five MROS3 homologues in A. thaliana, four of them arranged in two tandems, each consisting of two copies. We conclude that MROS3 is a low-copy gene family, connected with the proper pollen development, which is present not only in dioecious but also in other dicot plant species.

Amino Acid Sequence↗
PubMed2004

VOZ; isolation and characterization of novel vascular plant transcription factors with a one-zinc finger from Arabidopsis thaliana.

A 38-bp pollen-specific cis-acting region of the AVP1 gene is involved in the expression of the Arabidopsis thaliana V-PPase during pollen development. Here, we report the isolation and structural characterization of AtVOZ1 and AtVOZ2, novel transcription factors that bind to the 38-bp cis-acting region of A. thaliana V-PPase gene, AVP1. AtVOZ1 and AtVOZ2 show 53% amino acid sequence similarity. Homologs of AtVOZ1 and AtVOZ2 are found in various vascular plants as well as a moss, Physcomitrella patens. Promoter-beta-glucuronidase reporter analysis shows that AtVOZ1 is specifically expressed in the phloem tissue and AtVOZ2 is strongly expressed in the root. In vivo transient effector-reporter analysis in A. thaliana suspension-cultured cells demonstrates that AtVOZ1 and AtVOZ2 function as transcriptional activators in the Arabidopsis cell. Two conserved regions termed Domain-A and Domain-B were identified from an alignment of AtVOZ proteins and their homologs of O. sativa and P. patens. AtVOZ2 binds as a dimer to the specific palindromic sequence, GCGTNx7ACGC, with Domain-B, which is comprised of a functional novel zinc coordinating motif and a conserved basic region. Domain-B is shown to function as both the DNA-binding and the dimerization domains of AtVOZ2. From highly the conservative nature among all identified VOZ proteins, we conclude that Domain-B is responsible for the DNA binding and dimerization of all VOZ-family proteins and designate it as the VOZ-domain.

Arabidopsis↗
PubMed2004

Metabolic fate of jasmonates in tobacco bright yellow-2 cells.

Jasmonic acid and methyl jasmonate play an essential role in plant defense responses and pollen development. Their levels are temporarily and spatially controlled in plant tissue. However, whereas jasmonate biosynthesis is well studied, metabolic pathways downstream of jasmonic acid are less understood. We studied the uptake and metabolism of jasmonic acid and methyl jasmonate in tobacco (Nicotiana tabacum) Bright Yellow-2 suspension culture. We found that upon uptake, jasmonic acid was metabolized to its Glc and gentiobiose esters, and hydroxylation at C-11 or C-12 occurred. Free hydroxylated jasmonates were the preferential fraction of the culture medium. Upon hydrolysis of methyl jasmonate to jasmonic acid, a similar set of conversions occurs. In contrast to jasmonic acid, none of its derivatives interfere with the G2/M transition in synchronized tobacco Bright Yellow-2 cells.

Acetates↗
PubMed1979

Hysteresis in the responses of membrane potential, membrane resistance, and growth rate to cyclic temperature change.

Measurements of electrical potential, membrane resistance, and elongation rate have been carried out on the developing pollen tube of Oenothera drummondii.The plasmalemma potential was observed to be -138 millivolts +/- 19 (sd). Approximately 70% of this potential was apparently due to the operation of an electrogenic pump(s). KCN rapidly and reversibly depolarized the potential to about -40 millivolts. Lowering the temperature from 20 to 4 C brought about similar rapid depolarization. The effects of KCN and of temperature were in no degree additive. KCN had only a small effect on membrane resistance. In contrast, the latter was markedly increased by lowering the temperature.When the pollen tubes were submitted to cyclic temperature changes striking hysteresis effects were observed in the response of all three parameters, membrane potential, resistance, and growth rate. The hysteresis pattern for potential differed from that for resistance but resembled that for growth rate (measured simultaneously on the same pollen tube). The correlation coefficient between potential and growth rate was very high.The probable relevance to our results of the hysteresis phenomena associated with "clustering" and phase transition in lipids is pointed out. Attention is also drawn to the possible significance of the large difference between the electric potentials at the start of the cooling and heating paths, respectively.

Journal Article↗
PubMed1987

Plant factors controlling seed set in maize : the influence of silk, pollen, and ear-leaf water status and tassel heat treatment at pollination.

In maize (Zea mays L.) large decreases in kernel number result when low water potentials (Psi(w)) and high temperatures occur during pollination. To gain insight into the basis for the decreased seed set, silk, pollen, and ear-leaf Psi(w), the capability for silk osmotic adjustment, and pollen appearance were measured to determine their relationship to seed set. A multiple-eared or prolific (high carbohydrate availability to the pistillate inflorescence) hybrid (B73 x FR25), a heat sensitive hybrid (WF9 x A632), and a commercial hybrid (B73 x Mo17) were studied. A cross-pollination experiment, with pollination limited by pollen amount, was conducted to determine the impact on seed set of water and heat stressing the tassel and water stressing the ear. At low Psi(w), silk Psi(w) and seed set were decreased whereas pollen Psi(w), appearance, and viability were unaffected. High temperature resulted in a 2 megapascal decrease in pollen Psi(w), visually damaged pollen being shed, decreased pollen viability, and, in two of the hybrids, substantially decreased pollen shed. Prolificacy did not result in increased silk solute accumulation but did result in superior seed production by the pistillate inflorescence at low Psi(w). The magnitude of the decrease in silk solute potential was small (0.2 megapascal) and similar for all genotypes. One hybrid maintained a relatively high silk turgor but this hybrid also decreased the most in seed production when the pistillate inflorescence was water deficient. These results indicated an adverse effect of high temperature on pollen development, a positive relationship between seed production and silk water status, and no advantage to high silk turgor after silk emergence in maintaining seed production. Additionally, there was no evidence of variation in silk solute regulation capability among hybrids which varied in prolificacy, a trait important in drought tolerance, but the seed production of the pistillate inflorescence of the prolific hybrid was least affected by water deficit.

Journal Article↗
PubMed1995

Specific expression in reproductive tissues and fate of a mitochondrial sterility-associated protein in cytoplasmic male-sterile bean.

In common bean, cytoplasmic male sterility has been associated with a unique sequence found in the mitochondrial genome, designated pvs (for Phaseolus vulgaris sterility sequence). Within the pvs sequence, two open reading frames are encoded, ORF98 and ORF239. We have raised rabbit polyclonal antibodies against Pvs-ORF239 to evaluate the role of this putative male sterility-associated protein. Histological investigation of pollen development revealed that in the male-sterile bean line, callose deposition was abnormal and microspores remained as tetrads as previously reported. Pvs-ORF239 was found to be localized within the reproductive tissues of the male-sterile bean line, in contrast to all other cytoplasmic male sterility systems studied to date. This protein was associated with mitochondria, the callose layer, and developing primary cell walls during microsporogenesis. Expression of pvs-orf239 was not detected in fertile plants containing restorer gene Fr2. These observations, together with previous reports, suggest that nuclear restorer gene Fr2 interferes with expression of the pvs region post-transcriptionally.

Animals↗
PubMed2005

Transcriptional gene silencing as a tool for uncovering gene function in maize.

Transcriptional gene silencing has broad applications for studying gene function in planta. In maize, a large number of genes have been identified as tassel-preferred in their expression pattern, both by traditional genetic methods and by recent high-throughput expression profiling platforms. Approaches using RNA suppression may provide a rapid alternative means to identify genes directly related to pollen development in maize. The male fertility gene Ms45 and several anther-expressed genes of unknown function were used to evaluate the efficacy of generating male-sterile plants by transcriptional gene silencing. A high frequency of male-sterile plants was obtained by constitutively expressing inverted repeats (IR) of the Ms45 promoter. These sterile plants lacked MS45 mRNA due to transcriptional inactivity of the target promoter. Moreover, fertility was restored to these promoter IR-containing plants by expressing the Ms45 coding region using heterologous promoters. Transcriptional silencing of other anther-expressed genes also significantly affected male fertility phenotypes and led to increased methylation of the target promoter DNA sequences. These studies provide evidence of disruption of gene activity in monocots by RNA interference constructs directed against either native or transformed promoter regions. This approach not only enables the correlation of monocot anther-expressed genes with functions that are important for reproduction in maize, but may also provide a tool for studying gene function and identifying regulatory components unique to transcriptional gene control.

Fertility↗
PubMed2003

Characterization of rice functional monosaccharide transporter, OsMST5.

cDNA of a monosaccharide transporter in rice, OsMST5 (Oryza sativa monosaccharide transporter 5) was cloned and its sugar transport activity was characterized by heterologous expression analysis. The amino acid sequence and topology were similar to the sequences and topology of other plant monosaccharide transporters. Yeast cells co-expressed with OsMST5 cDNA transported some monosaccharide substrates. The transport rate increased when ethanol as an electron donor was added, so the transporter was an energy-dependent active one. Most of the OsMST5 was expressed in panicles before pollination, indicating that it is associated with pollen development in rice.

Amino Acid Sequence↗
PubMed1992

Titration of immunotherapy by periodical nasal allergic challenges in the treatment of allergic rhinitis.

OBJECTIVE: To determine the benefits of nasal allergen challenge (NAC) in monitoring immunotherapy for allergic rhinitis in a clinical setting. DESIGN: Two hundred consecutive courses of immunotherapy, with pyridine-extracted alum-precipitated allergen extracts (Allpyral), were personally carried out by the writer and analysed. Diagnosis before treatment was confirmed by NAC and the duration of treatment determined by periodical NAC. SETTING: Consultant allergist's clinical practice. PARTICIPANTS: Patients with allergic rhinitis who were referred for treatment; 79 patients were allergic to grass, 70 to the house dust mite, 30 to plantain and 21 to miscellaneous allergens. RESULTS: The mean protein nitrogen units (pnu) required to reverse an NAC was 145 467 pnu for grass, 93 771 pnu for mite, 103 137 pnu for plantain, and 121 030 pnu for miscellaneous allergen patients. The range of injections to obtain conversion to a negative NAC was wide. For grass it was 9-58 injections, for mite 6-47 injections, for plantain 4-39 injections, and for miscellaneous allergens 10-79 injections. Ten grass-sensitive patients who received an average of 477 807 pnu failed to convert to a negative NAC result. Similarly, two mite-sensitive patients who received 467 370 PNU, and one plantain-sensitive patient, one cat-sensitive patient and one horse-sensitive patient, who had 389 240 pnu, 148 520 pnu and 719 540 pnu respectively, failed to convert. One patient who was very sensitive to grass pollen developed urticaria which required an injection of adrenaline. CONCLUSION: Immunotherapy for allergic rhinitis with Allpyral is effective--as determined by the conversion of NAC results from positive to negative--providing a sufficient dose is given. The manufacturer's recommended dose is inadequate. The procedure is safe for mite, plantain and miscellaneous allergens, and only one reaction occurred with grass given in a high dosage protocol, which has subsequently been altered. Considering that the British Committee of Safety of Medicines reported no deaths from Allpyral injections, its recommendations appear over cautious for this make of extract.

Allergens↗
PubMed2006

[Identification of the rice (Oryza sativa L.) mutant msp1-4 and expression analysis of its UDT1 and GAMYB genes].

A rice male-sterile mutant msp1-4 (MULTIPLE SPOROCYTE) with japonica cultivar '9522' background, was obtained in M(3) population treated with (60)Co gamma-ray. Results of genetic analysis indicated that the male-sterile phenotype was controlled by a single recessive locus. To map this locus, an F(2) population was constructed from the cross between the msp1-4 (japonica) and 'LongTeFu B' (indica). This locus was mapped between the two InDel markers, WY-4 and WY-8, with physical distance of 247 kb. A deletion with 10 base pairs between 758 bp and 767 bp in MSP1 open reading frame was confirmed by sequence analysis, which led to pre-termination of MSP1 translation. Phenotype analysis of msp1-4 indicated that it was similar to the msp1 mutant. To insight the expression change of rice anther developmental genes in this mutant, semi-quantitative RT-PCR analysis was carried out. The results showed that the expression level of rice UDT1 and GAMYB were reduced in msp1-4, implying that UDT1 and GAMYB are possibly the downstream genes of MSP1 gene in rice pollen development.

Flowers↗
PubMed1995

Mitochondrial citrate synthase from potato: predominant expression in mature leaves and young flower buds.

A cDNA clone encoding mitochondrial citrate synthase (EC 4.1.3.7), the first enzyme of the tricarboxylic-acid cycle, was isolated from potato (Solanum tuberosum L.) and expression of the enzyme analyzed. The deduced amino-acid sequence of the potato mitochondrial citrate synthase showed high similarity to known citrate synthases from fungi, mammals and Arabidopsis thaliana. The expression pattern of this clone was determined by Northern blot analysis. Expression was detected in all tissues analyzed. The highest level of expression was found in green flower buds. In photosynthetic tissues, stronger mRNA expression was detected in mature than in immature leaves. This rise in expression with leaf age was accompanied by an increase in citrate-synthase activity. Within flowers, expression was severalfold stronger in anthers than in ovaries, indicating a role of mitochondrial citrate synthase during anther or pollen development. A comparatively low level of transcript was detected in underground heterotrophic tissues, such as stolons, tubers and roots. When tubers were stored at low temperature (4 degrees C), mitochondrial citrate-synthase gene expression increased slightly. From the data obtained, we conclude that expression of the mitochondrial citrate-synthase gene is regulated by developmental and environmental factors. The relatively high expression in leaves is in line with the assumption that mitochondria play an important role in photosynthetically active tissues.

Amino Acid Sequence↗
PubMed1993

[Hyposensitization treatment of allergic diseases].

Hyposensitization or allergenic immunotherapy has been used extensively for 80 years as a specific treatment of IgE-mediated allergic diseases. Highly effective is venom immunotherapy for patients with systemic reactions after hymenoptera stings and positive diagnostic tests for venom-specific IgE. The clinical efficacy of immunotherapy in hay fever has also been convincingly documented. 20 to 30% of patients with allergic rhinitis pollen develop asthma at a later stage of the disease. Some studies have shown a significant efficacy of immunotherapy in reducing the further development of rhinitis into asthma. The clinical efficacy of immunotherapy in allergic asthma to house-dust mites, animal danders or moulds has been questioned; however, a number of controlled studies published during the last decade shows a clear tendency towards a beneficial effect, especially in children. Recent data show a reduction in mediator release induced by immunotherapy and an inhibition of the late-phase reaction following skin and bronchial challenge tests. Immunotherapy and pharmacotherapy should be integrated components of the treatment strategy of allergic diseases in order to gain the optimal result for the allergic patient.

Desensitization, Immunologic↗