Disruption of apyrases inhibits pollen germination in Arabidopsis

In Arabidopsis, we previously identified two highly similar apyrases, AtAPY1 and AtAPY2. Here, T-DNA knockout (KO) mutations of each gene were isolated in a reverse genetic approach. The single KO mutants lacked a discernible phenotype. The double KO mutants, however, exhibited a complete inhibition of pollen germination, and this correlated with positive beta-glucuronidase staining in the pollen of apyrase promoter:beta-glucuronidase fusion transgenic lines. The vast majority of the pollen grains of these mutants were identical to wild type in size, shape, and nuclear state and were viable as assayed by metabolic activity and plasma membrane integrity. Complementation with either AtAPY1 or AtAPY2 cDNA rescued pollen germination, confirming that the phenotype was apyrase specific. Despite the redundancy of the two apyrases in rescue potential, transmission analyses suggested a greater role for AtAPY2 in male gamete success. The effect of mutant apyrase on the transmission through the female gametophyte was only marginal, and embryo development appeared normal in the absence of apyrases. The male-specific double KO mutation is fully penetrant and shows that apyrases play a crucial role in pollen germination.     

Apyrase from pea stems: Isolation, purification, characterization and identification of a NTPase from the cytoskeleton fraction of pea stem tissue

The cytoskeleton pellet from the first internode of dark-grown pea stems was disintegrated in a high salt buffer, ultracentrifuged to remove ribosomes and the post-ribosomal supernatant was applied to a heparin affinity column. Significant ATPase activity was present in the cytoskeleton fraction and this was eluted from the column at 0.6–0.7 M KOAc, in the same fractions as a 49-kDa protein (which we called B3). B3 was desalted and further purified by cation exchange column chromatography. Purified B3 catalyzed hydrolysis of ATP, CTP, GTP, TTP, UTP and ADP and thus appears to be an apyrase (ATP diphosphohydrolase, EC 3.6.1.5). Partial amino acid sequences of three major fragments were obtained by digestion of B3 by Staphylococcus aureus V8 protease (EC 3.4.21.19), and all these sequences were consistent with the previously reported amino acid sequences for pea nucleoside triphosphatase (NTPase, EC 3.6.1.15) (PIR S48859), which is thought to be an apyrase.     

Accumulation of Vacuolar H+-Pyrophosphatase and H+-ATPase during Reformation of the Central Vacuole in Germinating Pumpkin Seeds

Protein storage vacuoles were examined for the induction of H+-pyrophosphatase (H+-PPase), H+-ATPase, and a membrane integral protein of 23 kD after seed germination. Membranes of protein storage vacuoles were prepared from dry seeds and etiolated cotyledons of pumpkin (Cucurbita sp.). Membrane vesicles from etiolated cotyledons had ATP- and pyrophosphate-dependent H+-transport activities. H+-ATPase activity was sensitive to nitrate and bafilomycin, and H+-PPase activity was stimulated by potassium ion and inhibited by dicyclohexylcarbodiimide. The activities of both enzymes increased after seed germination. On immunoblot analysis, the 73-kD polypeptide of H+-PPase and the two major subunits, 68 and 57 kD, of vacuolar H+-ATPase were detected in the vacuolar membranes of cotyledons, and the levels of the subunits of enzymes increased parallel to those of enzyme activities. Small amounts of the subunits of the enzymes were detected in dry cotyledons. Immunocytochemical analysis of the cotyledonous cells with anti-H+-PPase showed the close association of H+-PPase to the membranes of protein storage vacuoles. In endosperms of castor bean (Ricinus communis), both enzymes and their subunits increased after germination. Furthermore, the vacuolar membranes from etiolated cotyledons of pumpkin had a polypeptide that cross-reacted with antibody against a 23-kD membrane protein of radish vacuole, VM23, but the membranes of dry cotyledons did not. The results from this study suggest that H+-ATPase, H+-PPase, and VM23 are expressed and accumulated in the membranes of protein storage vacuoles after seed germination. Overall, the findings indicate that the membranes of protein storage vacuoles are transformed into those of central vacuoles during the growth of seedlings.     

On the Composition, Deposition and Mobilization of Proteins in the Cotyledons of Castor Bean (Ricinus communis L. cv. Hale) Seeds: Their Role as Storage Proteins

Proteins in the soluble and insoluble fractions, extracted frommature castor bean cv. Hale seed cotyledons, differ quantitativelyand qualitatively from their counterparts extracted from theendosperm. The soluble fraction contains no glycoproteins, andthe lectins RCA₁ and ricin D are absent. While the insolubleproteins are electrophoretically and immunologically similarto those in the endosperm, they do not form the 100 kD subunitdimers which characterize some of the endosperm insoluble crystalloidproteins. Rapid rates of deposition of all of the soluble andinsoluble proteins present in the mature seed cotyledons commences30–35 d after pollination (DAP) and continues until 45DAP. These proteins are mobilized rapidly beginning 1–2d after seed imbibition and this coincides with an increasein specific activity, in the cotyledons, of two aminopeptidasesand a carboxypeptidase. The soluble and insoluble proteins inthe cotyledons of the mature seed probably function as storageproteins and support the growth of the germinated seed priorto the mobilization of the major protein storage reserves ofthe endosperm. Key words: Ricinus communis, Castor bean, Hale cultivar, Cotyledon, Storage protein, Seed development, Seed germination     

Differential expression of two soybean apyrases, one of which is an early nodulin

Two cDNA clones were isolated from soybean (Glycine soja) by polymerase chain reaction with primers designed to conserved motifs found in apyrases (nucleotide phosphohydrolase). The two cDNAs are predicted to encode for two, distinct, apyrase proteins of approximately 50 kDa (i.e., GS50) and 52 kDa (i.e., GS52). Phylogenetic analysis indicated that GS52 is orthologous to a family of apyrases recently suggested to play a role in legume nodulation. GS50 is paralogous to this family and, therefore, likely plays a different physiological role. Consistent with this analysis, GS50 mRNA was detected in root, hypocotyls, flowers, and stems, while GS52 mRNA was found in root and flowers. Neither gene was expressed in leaves or cotyledons. Inoculation of roots with Bradyrhizobium japonicum, nitrogen-fixing symbiont of soybean, resulted in the rapid (<6 h) induction of GS52 mRNA expression. The level of GS50 mRNA expression was not affected by bacterial inoculation. Western blot (immunoblot) analysis of GS50 expression mirrored the results obtained by mRNA analysis. However, in contrast to the mRNA results, GS52 protein was found in stems. Interestingly, anti-GS52 antibody recognized a 50-kDa protein found only in nodule extracts. Treatment of roots with anti-GS52 antibody, but not anti-GS50 antibody or preimmune serum, blocked nodulation by B. japonicum. Fractionation of cellular membranes in sucrose density gradients and subsequent Western analysis of the fractions revealed that GS50 colocalized with marker enzymes for the Golgi, while GS52 colocalized with marker enzymes for the plasma membrane. Restriction fragment length polymorphism (RFLP)-based mapping placed the gs52 gene on major linkage group J of the integrated genetic map of soybean. These data suggest that GS50 is likely an endo-apyrase involved in Golgi function, while GS52 is localized on the root surface and appears to play an important role in nodulation.     

Mitochondrial and Peroxisomal β-Oxidation of Stearic and Lignoceric Acids by Rat Brain

Crude subcellular fractions were prepared from adult rat brains by differential centrifugation of brain homogenates. Greater than 98% of the cellular mitochondrial marker enzyme activity sedimented in the heavy and light mitochondrial pellets, and less than 1% of the activity sedimented in microsomal pellets. Lysosomal marker enzyme activities mainly (71-78% of cellular activity) sedimented in the heavy and light mitochondrial pellets. Significant amounts of the lysosomal marker enzyme activity also sedimented in the crude microsomal pellets (9-13% of total) and high-speed supernatants (14-16% of total). The specific activities of microsomal and peroxisomal marker enzyme activities were highest in the crude microsomal pellets. Fractionation of the crude microsomal pellets on Nycodenz gradients resulted in the separation of the bulk of the remaining mitochondrial, lysosomal, and microsomal enzyme activities from peroxisomes. Fatty acyl-CoA synthetase activities separated on Nycodenz gradients as two distinct peaks, and the minor peak of the activities was in the peroxisomal enriched fraction. Fatty acid beta-oxidation activities also separated as two distinct peaks, and the activities were highest in the peroxisomal enriched fractions. Mitochondria were purified from the heavy mitochondrial pellets by Percoll density gradients. Fatty acyl-CoA synthetase and fatty acid beta-oxidation activities were present in both the purified mitochondrial and peroxisomal enriched fractions. Stearoyl-CoA synthetase activities were severalfold greater compared to lignoceroyl-CoA synthetase, and stearic acid beta-oxidation was severalfold greater compared to lignoceric acid beta-oxidation in purified mitochondrial and peroxisomal enriched fractions.(ABSTRACT TRUNCATED AT 250 WORDS)     

Maize seed cryo-storage modifies chlorophyll,carotenoid, protein,aldehyde and phenolics levels during early stages of germination

We recorded the crypreservation effects (direct immersion) on various parameters of early germination stages of maize seeds (0, 7 and 14 days). Percentages of germination; fresh mass of different seedling parts; levels of chlorophyll pigments (a, b); carotenoids; malondialdehyde; other aldehydes; phenolics (cell wall-linked, free) and proteins were determined. Various statistically significant effects of seed exposure to liquid nitrogen (LN) were recorded. Maize seeds did not seem to be affected by LN exposure either visually or regarding fresh weight or germination rate. However, delayed growth was observed in seedlings recovered from cryopreserved seeds. This trend indicated an increase in the effect of seed cryopreservation on growing plants. The most significant effects of LN exposure were recorded in the combined fresh weight of stems and leaves at day 7 of germination and in fresh weights of roots, stems and leaves at day 14. At the biochemical level, numerous indicators varied following LN exposure, but the most significant effects were recorded in carotenoids, malondialdehyde and other aldehyde contents. LN exposure modified 50.0% of indicators in cotyledons, 48.1% in stems and leaves, 38.8% in roots and 11.1% in seeds. LN storage modified 11.1% of the variables measured at day 0 of germination, 37.0% at day 7, and 52.7% at day 14. Field performance of cryostored seed-derived plants should be evaluated to measure the durability of the changes observed.     

Cowpea ribonuclease: properties and effect of NaCl-salinity on its activation during seed germination and seedling establishment

Pitiúba cowpea [Vigna unguiculata (L.) Walp] seeds were germinated in distilled water (control treatment) or in 100 mM NaCl solution (salt treatment), and RNase was purified from different parts of the seedlings. Seedling growth was reduced by the NaCl treatment. RNase activity was low in cotyledons of quiescent seeds, but the enzyme was activated during germination and seedling establishment. Salinity reduced cotyledon RNase activity, and this effect appeared to be due to a delay in its activation. The RNases from roots, stems, and leaves were immunologically identical to that found in cotyledons. Partially purified RNase fractions from the different parts of the seedling showed some activity with DNA as substrate. However, this DNA hydrolyzing activity was much lower than that of RNA hydrolyzing activity. The DNA hydrolyzing activity was strongly inhibited by Cu²⁺, Hg²⁺, and Zn²⁺ ions, stimulated by MgCl₂, and slowly inhibited by EDTA. This activity from the most purified fraction was inhibited by increasing concentrations of RNA in the reaction medium. It is suggested that the major biological role of this cotyledon RNase would be to hydrolyze seed storage RNA during germination and seedling establishment, and it was discussed that it might have a protective role against abiotic stress during later part of seedling establishment.     

莲种子萌发和幼苗生长时期营养物质的代谢变化

莲子叶细胞中储存了丰富的营养物质, 主要为蛋白质、淀粉和淀粉质体DNA。这些贮藏物质为种子萌发和幼苗的生长提供必需的能量和养料。通过组织化学和显微镜观察, 研究莲从种子萌发到植株生长至具有4个节时, 子叶中贮藏物质消耗的全过程。在此过程中, 子叶中的贮藏物质不断降解,营养物质发生转运。蛋白体首先发生降解, 其大量降解主要发生在幼苗三叶期。淀粉质体降解时会聚 集成团, 之后体积逐渐减小, 最后完全降解。种子萌发后65天是子叶贮藏物质消耗末期, 淀粉质体DNA的含量比萌发后20天的三叶期明显减少。细胞壁的形态结构发生多种形式的变化, 细胞壁发生的这些变化与子叶细胞间物质的运输有关。含多糖的球形颗粒通过维管束在子叶中运输。     

莲种子萌发和幼苗生长时期营养物质的代谢变化

莲子叶细胞中储存了丰富的营养物质,主要为蛋白质、淀粉和淀粉质体DNA.这些贮藏物质为种子萌发和幼苗的生长提供必需的能量和养料.通过组织化学和显微镜观察,研究莲从种子萌发到植株生长至具有4个节时,子叶中贮藏物质消耗的全过程.在此过程中,子叶中的贮藏物质不断降解,营养物质发生转运.蛋白体首先发生降解,其大量降解主要发生在幼苗三叶期.淀粉质体降解时会聚集成团,之后体积逐渐减小,最后完全降解.种子萌发后65天是子叶贮藏物质消耗末期,淀粉质体DNA的含量比萌发后20天的三叶期明显减少.细胞壁的形态结构发生多种形式的变化,细胞壁发生的这些变化与子叶细胞间物质的运输有关.含多糖的球形颗粒通过维管束在子叶中运输.     

Characterization of water distribution and activities of enzymes during germination in magnetically-exposed maize (Zea mays L) seeds

Magnetic seed treatment is one of the physical pre-sowing seed treatments to enhance the performance of crop plants. In our earlier experiment, we found significant increase in germination and vigour characteristics of maize (Zea mays L.) seeds subjected to magnetic fields. Among various combinations of magnetic field (MF) strength and duration, best results were obtained with MF of 100 mT for 2 h and 200 mT for 1 h exposure. The quicker germination in magnetically-exposed seeds might be due to greater activities of germination related enzymes, early hydration of membranes as well as greater molecular mobility of bulk and hydration water fractions. Thus, in the present study, changes in water uptake during imbibition and its distribution and activities of germinating enzymes during germination were investigated in maize seeds exposed to static magnetic fields of 100 and 200 mT for 2 and 1 h respectively by nuclear magnetic resonance (NMR) spectroscopy. The magnetically-exposed seed showed higher water uptake in phase II and III than unexposed seed. The longitudinal relaxation time T1 of seed water showed significantly higher values and hence greater molecular mobility of cellular water in magnetically-exposed seeds as compared to unexposed. Component analysis of T2 relaxation times revealed the early appearance of hydration water with least mobility and higher values of relaxation times of cytoplasmic bulk water and hydration water in magnetically-exposed over unexposed seeds. Activities of alpha-amylase, dehydorgenase and protease during germination were higher in magnetically-exposed seeds as compared to unexposed. The quicker germination in magnetically-exposed seeds might be due to greater activities of germination related enzymes, early hydration of membranes as well as greater molecular mobility of bulk and hydration water fractions.     

Purification and characteristics of functional properties of soluble nucleoside triphosphatase (apyrase) from bovine brain

Soluble NTPase, differing in its properties from known proteins exhibiting NTPase activity, was purified from bovine brain to homogeneity. The enzyme has pH optimum at 7.5 and shows absolute dependence on bivalent cations and broad substrate specificity towards nucleoside-5 -tri- and -diphosphates, characteristics of apyrases. The NTPase follows Michaelis-Menten kinetics in the range of investigated substrate concentrations, the apparent K(m) values for UTP, ITP, GTP, CTP, CDP, and ATP being 86, 25, 41, 150, 500, and 260 microM, respectively. According to gel-filtration and SDS-PAGE data, the molecular mass of the enzyme is 60 kD. The NTPase is localized in the cytosol fraction and expressed in different bovine organs and tissues. Total NTPase activity of extracts of bovine organs and tissues decreases in the following order: liver > heart > skeletal muscle > lung > brain > spleen > kidney ~ small intestine. The enzyme activity can be regulated by acetyl-CoA, alpha-ketoglutarate, and fructose-1,6-diphosphate acting as activators in physiological concentrations, whereas propionate exhibits an inhibitory effect.     

ATP hydrolyzing salivary enzymes of caterpillars suppress plant defenses

The oral secretions of herbivores are important recognition cues that can be used by plants to mediate induced defenses. In this study, a degradation of adenosine-5'-triphosphate (ATP) in tomato leaves was detected after treatment with Helicoverpa zea saliva. Correspondingly, a high level of ATPase activity in saliva was detected and three ATP hydrolyzing enzymes: apyrase, ATP synthase and ATPase 13A1 were identified in salivary glands. To determine the functions of these proteins in mediating defenses, they were cloned from H. zea and expressed in Escherichia coli. By applying the purified expressed apyrase, ATP synthase or ATPase 13A1 to wounded tomato leaves, it was determined that these ATP hydrolyzing enzymes suppressed the defensive genes regulated by the jasmonic acid and ethylene pathways in tomato plant. Suppression of glandular trichome production was also observed after treatment. Blood-feeding arthropods employ 5'-nucleotidase family of apyrases to circumvent host responses and the H. zea apyrase, is also a member of this family. The comparatively high degree of sequence similarity of the H. zea salivary apyrase with mosquito apyrases suggests a broader evolutionary role for salivary apyrases than previously envisioned.     

Proteolytic activities in seeds ofVigna unguiculata (L.) Walp

Proteolytic activities were studied in cotyledons of germinated and mature dry seed and axis of mature dry seed ofVigna unguiculata (L.) Walp. cv. Seridó using the following substrates: hemoglobin (pH 3.5), casein (pH 6.0), LPA (pH 7.0), and BAPA (pH 7.6). During the germination period (six days) examined, the total activities of LPA-ase and BAPA-ase decreased progressively in cotyledons corresponding to protein depletion. Consequently, the specific activities of the above proteases remain practically constant. In the case of caseinase and hemoglobinase, total activities increased in the cotyledons up to the third day of germination. Thereafter there was a decrease in these activities (total) but an increase in specific activities. Higher values than in the cotyledons for all the activities were obtained with the axis of mature dry seed. A 25 to 50 % ammonium sulfate fraction of a buffered extract from mature dry seed was used as source of enzyme for all the substrates used. Sephadex G-100 chromatography of the 25 to 50 % (NH⁴)²SO⁴ fraction gave two main peaks which correspond to relative molecular mass of 100 000 and 60 000 and contained all the activities.     

芡实种子萌发期子叶传递细胞发育的初步研究

芡实种子萌发期,子叶吸收外胚乳中养分供萌发和幼苗发育,具有吸器的功能。在种子萌发过程中,子叶的部分表皮细胞发育为传递细胞。其壁内突的生长以外切向壁为多,形成壁内突的造壁物质主要由高尔基体合成,并由其溢出的囊泡运送的。     

Differential regulation of a family of apyrase genes from Medicago truncatula

Four putative apyrase genes were identified from the model legume Medicago truncatula. Two of the genes identified from M. truncatula (Mtapy1 and Mtapy4) are expressed in roots and are inducible within 3 h after inoculation with Sinorhizobium meliloti. The level of mRNA expression of the other two putative apyrases, Mtapy2 and Mtapy3, was unaffected by rhizobial inoculation. Screening of a bacterial artificial chromosome library of M. truncatula genomic DNA showed that Mtapy1, Mtapy3, and Mtapy4 are present on a single bacterial artificial chromosome clone. This apyrase cluster was mapped to linkage group seven. A syntenic region on soybean linkage group J was found to contain at least two apyrase genes. Screening of nodulation deficient mutants of M. truncatula revealed that two such mutants do not express apyrases to any detectable level. The data suggest a role for apyrases early in the nodulation response before the involvement of root cortical cell division leading to the nodule structure.     

Sub-cellular distribution and isotypes of a 49-kDa apyrase from Pisum sativum

We isolated a 49-kDa protein from various sub-cellular fractions from pea (Pisum sativum L. var. Alaska) stems using heparin affinity and cation exchange column chromatography. The corresponding proteins from all these fractions were identified as apyrase (EC 3.6.1.5) because they hydrolyzed both nucleoside tri- and diphosphates into their respective monophosphates. Using an antibody raised against apyrase, we studied the enzyme’s sub-cellular distribution in isolated fractions and found significant amounts in the cell wall (50%), the supernatant (33%), the cytoskeleton (14%), and the nuclei (3%). Immuno-electron microscopy using gold-labeled antibody confirmed that apyrase was present in cell walls, nuclei, and in filamentous structures in the cytoplasm associated with ribosomes. Even though there is only one gene (with two alleles), for this protein, 2D gels indicated there were at least five isotypes, three being major, and the relative abundance of these isotypes differed in different fractions. Enzymes from all fractions: (a) hydrolyzed nucleoside triphosphates and diphosphates, but not monophosphates, (b) were insensitive to most ATPase inhibitors (azide, fluoride, nitrate, molybdate, ouabain, quercetin), but (c) were all inhibited by vanadium pentoxide at relatively high concentrations. There were, however, some subtle differences between enzymes from different sub-cellular fractions, including different ADP/ATP hydrolysis ratios. These results show that the 49-kDa apyrase is located in various compartments within the cell (cell wall, nuclei, and the cytoskeleton) and that the enzymes from all fractions are basically similar in their apyrase function. We suggest that the enzyme is modified in various ways to furnish different forms with different (non-apyrase) functions in different sub-cellular locations.     

Soybean lectin and related proteins in seeds and roots of le and le soybean varieties

The localizations of soybean lectin (SBL) and antigenically related proteins in cotyledons and roots of lectin positive (Le⁺) and lectin negative (Le⁻) soybean cultivars were compared by light level immunocytochemistry using antibodies produced against the 120 kilodalton (kD) native seed lectin tetramer or its subunits. Lectin is present in the protein bodies of cotyledons cells as are two other seed proteins, the Kunitz trypsin inhibitor and the storage protein glycinin. Analysis of single seed extracts by immunoblotting of sodium dodecyl sulfate-polyacrylamide gels using the same antibodies, reveals up to 4 milligrams of the 30 kD seed lectin protein is present per seed in the Le⁺ varieties. There is no detectable lectin in the protein bodies of Le⁻ cotyledons as determined by immunocytochemistry and immunoblotting. Enzyme-linked immunosorbent assay confirmed this result to a sensitivity of less than 20 nanograms per seed. In contrast, the roots of both Le⁺ and Le⁻ plants bind the seed lectin antibody during immunocytochemistry, with fluorescence mainly localized in vacuole-like bodies in the epidermis. Root extracts contain a 33 kD polypeptide that binds anti-SBL antibody at an estimated minimal level of 20 nanograms per 4-day seedling, or 2.0 nanograms per primary root tip. This polypeptide is also present in the embryo axis and in leaves. The latter also contain a 26 kD species that binds seed lectin antibody. The 30 kD seed lectin subunit, however, is not detectable in roots or leaves.     

Endophytic fungi from flowers, capsules and seeds of Eucalyptus globulus

The main goal of this work was to detect whether Cytospora chrysosperma and Fusicoccum eucalypti are present as endophytes of symptomless hypocotyls, cotyledons, flowers, capsules, peduncles of flowers in order to interpret an earlier finding of their presence in seeds, seedling stems and twigs of E. globulus. Segments from these organs as well as from bark and the xylem from flower peduncles were surface-sterilized and plated on 2% malt-agar. All plates were incubated at 24 degrees C for six weeks or more depending on the growth rate of fungi. C. chrysosperma was asymptomatically present in flowers, capsules, hypocotyls, cotyledons and peduncles. F. eucalypti was isolated from asymptomatic flowers and capsules. It is probable that C. chrysosperma spreads during seed germination colonizing seedling stems through hypocotyl and cotyledon.     

The Relation of Storage Protein to Vigor and Its Mobilization Pattern in Germinating Peanut Seeds

The peanut (Arachis hypogaea L.) seeds harvested at the last stage of maturation were divided into five grades by size. The content of total protein, salt-soluble protein, arachin, conarachin I and 2s globulin in these seeds were measured. No obvious differences in germination percentage and the length of radicle and hypocotyl within 3d germination in dark were observed among the five grades of seeds. But there were significant differences in the seedling growth after two weeks of germination in light. There was a very close correlation between the storage protein in cotyledons and the seedling growth. When seeds germinated in light, the efficiency of mobilization of the salt-soluble protein in the cotyledons was higher than that in the cotyledons of the seeds germinating in dark. All of the salt-soluble protein in cotyledons was used up after 14d seedling growth in light. SDS-PAGE of salt-soluble protein showed that 23.5, 38.5 and 41 kD subunits of arachin were first mobilized during germination. The 18 kD subunits of arachin were not mobilized until the above-mentioned subunits were used up. The 60.5 kD subunit of conarachin I and 2s globulin were degradated within 2 to 3 days during germination.