Proteolytic activity in the maize pollen wall

A new protease from maize ( Zea mays L.) pollen is described. It was purified using gel filtration, ion exchange and high performance liquid chromatography. SDS-PAGE and HPLC showed that the enzyme has a dimeric structure of M, ca 60,000. Inhibitor investigations indicated an aspartic acid residue in its active site. The optimum pH for maize pollen aspartic proteinase activity was 5.6, and the optimum temperature was 45°C. The enzyme is easily eluted from the pollen grains and, as confirmed by enzymoblotting after isoelectric focusing, it is located in the pollen wall. Similar to metallo-proteinases, its activity is inhibited by Zn₂₊. The pL value for purified aspartic proteinase, as estimated after IEF, was 5.0. Two-dimensional electrophoresis analysis of proteins eluted from maize pistils suggests that the enzyme digests the proteins and may be involved in pollen-tube germination. The properties of serine and aspartic proteinases from maize pollen are compared.     

Treatment of maize pollen to reduce nuclease activity

Recently it has been reported that maize (Zea mays L.) pollen can be stored up to several hours in a hypertonic aqueous medium at 0°C without losing its germinability (Broglia and Brunori 1994). We found that both release and activity of pollen nucleases are diminished in a cold hypertonic aqueous medium. Nucleases can be washed off while preserving germination ability and thus preserving the possibility of passive uptake of exogeneous DNA into germinating pollen grains. Alternatively, active DNA transfer into non-germinating pollen grains in the storage medium itself may be facilitated, due to the very reduced nuclease activity in this medium.     

Inhibition of the activity of aminoacyl-tRNA synthetases in pollen grains of maize

Extracts from pollen grains of maize ( Zea mays L.) show a low activity of aminoacyltRNA synthetases (EC 6. 1. 1). They also contain a specific factor inhibiting the activity of these enzymes. The molecular mass of this factor, which may be a short peptide, is about 3000 Da as determined by column chromatography on Sephadex G-25 Fine. The Michaelis constant (K_m), determined for the amino acid in the presence of this factor, suggests its allosteric influence on the affinity of the enzyme. Short-term incubation of the factor with pronase R resulted in conversion of the inhibiting action into a stimulating one. Kinetics of aminoacylation reactions confirm inhibitory and stimulative influences of the effector on the enzyme activity. High performance liquid chromatography shows that inhibition of the activity of aminoacyl-tRNA synthetases is affected by a group of compounds of similar molecular masses.     

Maturation of maize pollen in vitro

Summary Maturation of maize pollen was obtained in male reproductive structures cultured in vitro. Immature tassels containing microspores at the mid-uninucleate to late-binucleate stage of development were excised and spikelets, anthers, and/or isolated microspores were cultured on a medium capable of supporting pollen maturation. Microspore mitosis, culminating in the production of starch-filled, trinucleate pollen capable of germination, was observed after 7–15 days, depending on the genotype and stage at which the cultures were initiated. Up to 100%, 70%, and 20% of the cultured spikelets, anthers, and isolated microspores, respectively, produced mature pollen, which germinated, however, at different frequencies (i.e., spikelets, 50–70%; anthers, 5–10%; microspores, <1%). Mature kernels were produced following fertilization with pollen from cultured spikelets and anthers. These procedures provide methods for the in vitro manipulation of a significant phase of the maize life cycle.     

Glucosyltransferase activity of commercial juice-processing enzyme preparations

Of various commercial enzyme preparations examined, Cytolase M102 was found to contain the highest glucosyltransferase activity (55 U ml⁻¹). It rapidly converted maltose to panose (Glcα1 → 6Glcα1 → 4Glc) with a V _max value of 5·8 mmol l⁻¹ min⁻¹ at 50°C in 0·05 mol l⁻¹ sodium acetate buffer (pH 4·4). The K _m value of the enzyme for maltose was 750 mmol l⁻¹. Yields of panose and glucose after 45 min of reaction, for example, were 47·2% and 52·8%, respectively, on the basis of the amount of maltose consumed.     

Glucosylation of quercetin by a maize pollen enzyme

A glucosyltransferase present in maize pollen catalyses the formation of isoquercitrin from quercetin and uridine 5′-diphosphoglucose. The reaction also required reduced sylfhydryl groups and was stimulated by addition of magnesium ions. Two pH optima were observed for the catalytic activity, a minor one at 6·2 and a major one at 8·2. The enzyme has a temperature optimum of 37° and can be inhibited by mercuric salts. Whole pollen readily served as the source of the enzyme in the reaction mixture.     

Manganese deficiency in maize affects pollen viability

Maize (Zea mays L. cv. G2) was grown with 0.55 mg L^–1 (sufficient), or 0.0055 mg L^–1 (deficient) manganese in sand. Manganese-deficient plants developed visible deficiency symptoms and showed poor tasseling and delayed anther development. Compared to Mn-sufficient plants, Mn-deficient plants produced fewer and smaller pollen grains with reduced cytoplasmic contents. Manganese deficiency reduced in vitro germination of pollen grains significantly. Ovule fertility was not significantly affected by Mn. But in Mn-deficient plants seed-setting and development was reduced significantly.     

Heat-shock proteins during pollen development in maize

In contrast to sporophytic tissues, mature pollen of higher plants does not synthesize the typical set of heat-shock proteins (HSPs) in response to a marked temperature upshift. Immature grains, however, seem able to do so, at least partially. We investigated the characteristics of HSP synthesis throughout the male gametophytic phase in maize and compared gametophytic and sporophytic heat-shock responses. One-dimensional Sodium dodecyl sulfate-polyacryl-amide gel electrophoresis technique (SDS-PAGE) of newly synthesized proteins revealed that immature pollen synthesizes HSPs, some of which are not induced in sporophytic tissues. The heat-shock response appeared to be related to microgametophytic developmental stages. The strongest response was found in uninucleate microspores: at this stage, in addition to the sporophytic 102, 84, 72, and 18 kD HSPs, three other polypeptides of 74, 56, and 46 kD were observed. In the binucleate and trinucleate stages, only a reduced synthesis of few HSPs could be induced, and differences between genotypes were observed. In germinating pollen, HSP synthesis was not induced under a voriety of heat-stress conditions; however, the consti-tutive synthesis of two polypeptides of the same molecular weight, 72 and 64 kD, as two HSPs was observed. The biological significance of these results is discussed.     

Glucosyltransferase activity in mitochondria. Biosynthesis of glucosyl-phosphoryl-dolichol in inner mitochondrial membranes

1. Inner mitochondrial membranes are able to transfer [14C]glucose from UDP-[14C]glucose onto dolichylmonophosphate. 2. Synthesis of dolichyl-phosphoryl-glucose takes place only in the presence of exogenous dolichyl-monophosphate loaded into phospholipid vesicles. 3. Neutral phospholipids interact preferentially with the membrane-bound enzyme. The effect of phospholipids is not related to the length of fatty acid chains but a correlation between the activation and the degree of unsaturation of fatty acid chains has been found. 4. This enzyme required divalent cations for activity. Such a requirement might be related to lipid-protein interactions which favour a suitable conformation of glycosyltransferase.     

A group-1 grass pollen allergen influences the outcome of pollen competition in maize

Worldwide, 400 million people suffer from hay fever and seasonal asthma. The major causative agents of these allergies are pollen specific proteins called the group-1 grass pollen allergens. Although details of their antigenicity have been studied for 40 years with an eye towards immunotherapy, their function in the plant has drawn scant attention. Zea m 1 constitutes a class of abundant grass pollen allergens coded for by several genes that loosen the walls of grass cells, including the maize stigma and style. We have examined the impact of a transposon insertion into one of these genes (EXPB1, the most abundant isoform of Zea m 1) on the production of Zea m 1 protein, pollen viability, and pollen tube growth, both in vitro and in vivo. We also examined the effect of the insertional mutation on the competitive ability of the pollen by experimentally varying the sizes of the pollen load deposited onto stigmas using pollen from heterozygous plants and then screening the progeny for the presence of the transposon using PCR. We found that the insertional mutation reduced the levels of Zea m 1 in maize pollen, but had no effect on pollen viability, in vitro pollen tube growth or the proportion of progeny sired when small pollen loads are deposited onto stigmas. However, when large pollen loads are deposited onto the stigmas, the transposon mutation is vastly underrepresented in the progeny, indicating that this major pollen allergen has a large effect on pollen tube growth rates in vivo, and plays an important role in determining the outcome of the pollen-pollen competition for access to the ovules. We propose that the extraordinary abundance (4% of the extractable protein in maize pollen) of this major pollen allergen is the result of selection for a trait that functions primarily in providing differential access to ovules.     

Clinical effects of a hop water extract on Japanese cedar pollinosis during the pollen season: a double-blind, placebo-controlled trial

The clinical effects of an oral administration of a hop water extract (HWE) on the improvement of Japanese cedar pollinosis (JCPsis) symptoms were investigated. In a double-blind, placebo-controlled trial, 39 subjects took a drink containing either 100 mg of HWE or a placebo for 12 weeks during the pollen season. Nasal symptoms (sneezing attacks, nasal discharge, and nasal obstruction) were assessed from the subjects' diaries. A clinical examination and blood sampling were carried out before and 4, 8 and 12 weeks after the initiation of treatment. As a result, a significant difference was observed in the symptom score and in the symptom-medication score 10 weeks after the intervention in comparison with the placebo group. Improvements were observed in nasal swelling, nasal color, amount of nasal discharge, and characteristics of nasal discharge in the intervention group 12 weeks after the treatment. No significant eosinophil infiltration into the nasal discharge was apparent in the intervention group throughout the study period, although it was observed in the placebo group. These findings indicate that an oral administration of HWE may be effective in alleviating the allergic symptoms related to JCPsis.     

Proteome analysis of maize pollen for allergy-relevant components

Over the last few decades, the cultivation of maize (Zea mays) has strongly increased in Central Europe. We therefore decided to study the allergen composition and the allergenic potency of its pollen in comparison with pollen from timothy grass (Phleum pratense), a typical representative of the native grasses. We found that 65% of the sera reactive to timothy pollen also bound to maize pollen proteins. By using 2-DE immunoblotting, followed by incubation with mAbs directed against known allergens or protein sequencing, those IgE-reactive components were further classified. Although novel, maize-specific pollen allergens could not be found, the presence of crossreacting allergens belonging to groups 1 and 13 (Zea m 1 and 13), both having high IgE prevalence, as well as the presence of the less important group 3 and 12 allergens was found. The structural variability of Zea m 1 and Zea m 13 was determined by sequencing clones isolated from a maize pollen cDNA library. This revealed sequence identities of 72 and 70%, respectively, to the corresponding Phl p 1 and Phl p 13 allergens of timothy grass pollen. IgE-crossreactivity was further studied using immunoblot inhibition tests. Here, timothy pollen extract completely blocked IgE binding to maize, whereas maize pollen extract blocked IgE reactivity to only some timothy pollen allergens.     

Antiviral activity of a hot water extract of black soybean against a human respiratory illness virus

Significant antiviral activity against respiratory illness viruses has been found in a hot-water extract of black soybean. This black soybean extract showed significant antiviral activity against human adenovirus type 1 and coxsackievirus B1 in a dose-dependent manner, while the hot-water extract from common yellowish soybean showed only weak activity. The antiviral activity could not be extracted from the black soybean by 70% aqueous ethanol, suggesting that saponin in the seed did not contribute to this activity. The antiviral activity was only recovered from cotyledons and not from seed coats with the hot water, showing that the activity was distributed in the cotyledons and that antocyanins in the black soybean seed coats did not contribute to the antiviral activity. The antiviral compound(s) in the black soybean was partially purified by up to 166 times by a combination of gel filtration, reversed phase HPLC, and ion-exchange HPLC. The partially purified antiviral compound showed hydrophilic and anionic properties, and a maximum absorption at 260 nm, suggesting that this antiviral fraction may contain a phenyl group(s). On the other hand, an amino acid analysis with the acid hydrolyzate and a neutral sugar analysis showed that the antiviral compound from black soybean might not be a polypeptide or glycoconjugate bearing neutral sugar(s).     

Competitive ability of maize pollen. Intergametophytic effects

Summary Intergametophytic influences on pollen competitive ability were studied by means of the pollen mixture technique: mixtures of pollen from different genetic sources, marked for the presence of the normal or the mutant allele of the opaque-2 gene, were used to pollinate o2o2 female plants. The variation of relative frequency of one of the two kernel types from apex to base of the resulting ear, made it possible to measure the competitive ability by regression coefficients.The effect of the pollen marker on the character was estimated by analyzing the progeny of heterozygous o2/+ plants self-pollinated through four generations. The o2 allele-bearing pollen revealed a slower germination rate than the dominant counterpart; no differences between the two pollen types were observed with regard to tube growth rate.The occurrence of intergametophytic interactions was confirmed: the competitive ability of a line varied according to the genotype of the competing pollen. Thus, this component has to be taken into account in considering male gametophyte fertilization ability.     

Purification and characterization of actin from maize pollen

Pollen is an excellent source of actin for biochemical and physiological studies of the actomyosin system in higher plants. We have developed an efficient method to prepare relatively high levels of actin from the pollen of maize (Zea mays L.). The procedures of purification include acetone powder preparation, saturated ammonium sulfate fractionation, diethylaminoethyl-cellulose chromatography, a cycle of polymerization-depolymerization, and Sephacryl S-200 gel filtration. The average yield of actin is 19 milligrams per 100 grams of pollen grains extracted. This is comparable with those of Acanthamoeba castellanii and human platelets. The purified pollen actin is electrophoretically homogeneous and its molecular mass is 42 kilodaltons. The amino acid composition and circular dichroism spectrum of pollen actin are identical to those of muscle actin. The actin purified from pollen is able to polymerize to F-actin. The pollen F-actin activated the activity of the muscle myosin ATPase sevenfold.     

Immature maize spikelets develop and produce pollen in culture

Immature maize spikelets have been successfully grown in vitro. Culture conditions were refined to maximize development of normal pollen grains. Kinetin was not required for normal development, in contrast to the absolute requirement for this plant growth regulator for in vitro tassel development. Development occured in all stages sampled, from premeiosis to postvacuolation, and there was no lag in progression through the various stages of development as compared to greenhouse-grown material. Cultured spikelets produced pollen that appeared morphologically normal, accumulated starch and had the normal two sperm nuclei and single vegetative nucleus.     

Meiotic chromosomal variation resulting from irradiation of pollen in maize

The objective of this study was to standardize an induction strategy of chromosome aberrations in maize inbred line L-869. Pollen grains irradiated with 0, 36 and 72 Gy were used for fertilization. Resulting seeds were planted in a greenhouse to assess the number of abnormal meiotic cells. Germination, height, sterility and mortality were verified. Cells with delayed separation of chromosomes, translocation, deficiency, abnormal pairing, later condensation and anaphase bridges were observed. The number of abnormalities increased as the dosage increased but chromosome aberration types were the same regardless of the dosages used. Various chromosome-altered plants were obtained without viability loss