Microsporogenesis in inbred line of popcorn (Zea mays L.)

Endogamy places genes for several characteristics in homozygosis, which include those related to meiosis causing abnormalities that may impair gamete viability. An original population (S0) of popcorn (CMS-43) produced by Embrapa Maize and Sorghum was self-pollinated for seven years, generating inbred lines (S1 to S7). Conventional studies of microsporogenesis revealed that meiotic abnormalities did not increase with endogamy. Univalent chromosomes, irregular chromosome segregation, abnormal cell shape, partial asynapsis, cell fusion, absence of cytokinesis, abnormal spindle orientation, and chromosome stickiness were recorded in low frequency in meiocytes. Since the frequency of abnormalities was low, mainly in S7, inbred lines from CMS-43 have a high potential for hybridization.     

Molecular diversity of arbuscular mycorrhizal fungi colonising Hyacinthoides non-scripta (bluebell) in a seminatural woodland

Arbuscular mycorrhizal (AM) fungi form symbiotic associations with plant roots. Around 150 species have been described and it is becoming clear that many of these species have different functional properties. The species diversity of AM fungi actively growing in roots is therefore an important component of ecosystem diversity. However, it is difficult to identify AM fungi below the genus level from morphology in planta , as they possess few informative characters. We present here a molecular method for identifying infrageneric sequence types that estimate the taxonomic diversity of AM fungi present in actively growing roots. Bluebell roots were sampled from beneath two different canopy types, oak and sycamore, and DNA sequences were amplified from roots by the polymerase chain reaction with fungal-specific primers for part of the small subunit ribosomal RNA gene. Restriction fragment length polymorphism among 141 clones was assessed and 62 clones were sequenced. When aligned, discrete sequence groups emerged that cluster into the three families of AM fungi: Acaulosporaceae, Gigasporaceae and Glomaceae. The sequence variation is consistent with rRNA secondary structure. The same sequence types were found at both sampling times. Frequencies of Scutellospora increased in December, and Acaulospora increased in abundance in July. Sites with a sycamore canopy show a reduced abundance of Acaulospora , and those with oak showed a reduced abundance of Glomus . These distribution patterns are consistent with previous morphological studies carried out in this woodland. The molecular method provides an alternative method of estimating the distribution and abundance of AM fungi, and has the potential to provide greater resolution at the infrageneric level.     

Microsporogenesis and tapetal development in fertile and cytoplasmic male-sterile sugar beet (Beta vulgaris L.)

Summary The development of sporogenous and tapetal cells in the anthers of male-fertile and cytoplasmic male-sterile sugar beet (Beta vulgaris L.) plants was studied using light and transmission electron microscopy. In general, male-sterile anthers showed a much greater variability in developmental pattern than male-fertile anthers. The earliest deviation from normal anther development was observed to occur in sterile anthers at meiotic early prophase: there was a degeneration or irregular proliferation of the tapetal cells. Other early aberrant events were the occurrence of numerous small vesicles in the microspore mother cells (MMC) and a disorganized chromatin condensation. Deviations that occurred in sterile anthers at later developmental stages included: (1) less distinct inner structures in the mitochondria of both MMC and tapetal cells from middle prophase onwards. (2) dilated ER and nuclear membranes at MMC prophase, in some cases associated with the formation of protein bodies. (3) breakdown of cell walls in MMCs and tapetal cells at late meiotic prophase. (4) no massive increase in tapetal ER at the tetrad stage. (5) a general dissolution of membranes, first in the MMC, then in the tapetum. (6) abortion of microspores and the occurrence of a plasmodial tapetum in anthers reaching the microspore stage. (7) no distinct degeneration of tapetal cells after microspore formation. Thus, it seems that the factors that lead to abortive microsporogenesis are structurally expressed at widely different times during anther development. Aberrant patterns are not restricted to the tetrad stage but occur at early prophase.     

T-URF 13 Protein from Mitochondria of Texas Male-Sterile Maize (Zea mays L.) : Its Purification and Submitochondrial Localization, and Immunogold Labeling in Anther Tapetum during Microsporogenesis

The protein T-URF13 (URF13) is specific to mitochondria of maize (Zea mays L.) with Texas (T) male-sterile cytoplasm and has been implicated in causing male sterility and susceptibility to T-cytoplasm-specific fungal diseases. T-URF13 was purified from isolated mitochondria from maize (line B73) with T cytoplasm by gel filtration and a quasi two-dimensional polyacrylamide gel electrophoresis system. Antibodies to the purified and denatured protein were produced in rabbits. Anti-T-URF13 antiserum was used to show that T-URF13 is in the inner membrane of mitochondria and behaves as an integral membrane protein when mitochondria are fractionated with sodium carbonate or Triton X-114. The antiserum and protein A tagged with 20-nanometer-gold particles were used to localize T-URF13 in T mitochondria by electron microscopy of sections of isolated mitochondria from etiolated shoots and sections of roots and of tapetal cells at pre-and post-degeneration stages of microsporogenesis. The microscopic study confirms that T-URF13 is specifically localized in the mitochondrial membranes of all of the T mitochondria tested, notably those in the tapetum from the meiocyte stage to the late-microspore stage. No change in the amount of labeled T-URF13 protein in the mitochondria of aging tapetal cells was detected.     

Ultrastructural Modifications of Plastids and Starch Metabolism during the Microsporogenesis of Ophrys lutea (Orchidaceae)

Ultrastructure of plastids has been studied throughout microsporogenesisof Ophrys lutea Cav. (Orchidaceae). A typical dedifferentiation/redifferentiationcycle of plastids was observed. At prophase I, plastids of pre-meiocytesgradually lost starch and separated the meiocytes through cytomicticchannels. At meiosis, plastids dedifferentiated to protoplastsand redifferentiated in young microspores. During pollen grainformation and maturation a cycle of starch accumulation/degradationoccurred; the mature pollen grain is starchless. Results arediscussed in terms of genetic inheritance following haploidnuclear state and in terms of carbohydrate metabolism. Plastids, starch, microsporogenesis, ultrastructure, Ophrys lutea, Orchidaceae     

Purification and Characterization of Porin from Corn (Zea mays L.) Mitochondria

Mitochondrial porin from corn (Zea mays L. B 73) shoots was solubilized with lauryl(dimethyl)-amine oxide and purified by chromatography on a hydroxyapatite:celite column. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the purified protein had an apparent molecular mass of 35 kD. When reconstituted in planar lipid bilayer membranes the porin formed ion-permeable channels with single-channel conductance of 2.0 and 4.0 nanosiemens in 1 M KCl. At low transmembrane voltages corn porin had the properties of a general diffusion pore with an estimated effective diameter of 1.6 nm and a small selectivity for anions over cations. The primary structure of corn porin seems to be quite different from that of other mitochondrial porins, because it did not cross-react with monoclonal antibodies against human porin and with polyclonal antibodies against yeast porin. Furthermore, the peptide maps of corn and bovine heart porins were very different. A sequence of 21 amino acids obtained by Edman degradation of peptides generated by porin proteolysis with Staphylococcus aureus V8 protease did not show any significant homology with known sequences of mitochondrial porins. Results of our investigation suggest that corn porin possesses functional properties similar to those of other mitochondrial porins, despite major structural differences.     

Protein Synthesis in Isolated Mitochondria of Rice (Oryza sativa L.) Seedlings

For studies of in organello mitochondrial protein synthesis in rice, Oryza sativa L., conventional surface-sterilization procedures were demonstrated to be ineffective. Because of the over-whelmingly efficient [³⁵S]methionine utilization by contaminating bacteria, even “essentially bacteria-free” rice mitochondria were shown to be unsuitable for the study of in organello protein synthesis. We developed a procedure to obtain a bacteria-free preparation of rice mitochondria. Such mitochondria favored a membrane-dependent ATP-generating system over an external ATP-generating system as the energy supplement for in organello protein synthesis. Two distinct classes of [³⁵S]methionine-labeled, cycloheximide-insensitive products were detected: an electrophoretically unresolved population and a set of some 22 to 27 discrete polypeptide species, each with a characteristic electrophoretic mobility and relative abundance.     

Mitochondria. II. The nuclear-mitochondrial relationship in Pelomyxa carolinensis Wilson (Chaos chaos L.)

This study has demonstrated that in the ameba, Pelomyxa carolinensis Wilson (Chaos chaos L.) the limiting membranes of mitochondria and postdivision nuclei are often continuous. The morphological relationship may be functional in that it permits an exchange of material resulting directly or indirectly in an increased enzyme content of the mitochondria. It is suggested that through a series of progressive foldings of its envelope, the nucleus may be a site of formation of mitochondria.     

Mitochondria : II. The Nuclear-Mitochondrial Relationship in Pelomyxa carolinensis Wilson (Chaos chaos L.)

This study has demonstrated that in the ameba, Pelomyxa carolinensis Wilson (Chaos chaos L.) the limiting membranes of mitochondria and postdivision nuclei are often continuous. The morphological relationship may be functional in that it permits an exchange of material resulting directly or indirectly in an increased enzyme content of the mitochondria. It is suggested that through a series of progressive foldings of its envelope, the nucleus may be a site of formation of mitochondria.     

Solubilization of a Proline Dehydrogenase from Maize (Zea mays L.) Mitochondria

L-Proline is oxidized to pyrroline-5-carboxylic acid in intact plant mitochondria by a proline dehydrogenase (EC 1.4.3) that is bound to the matrix side of the inner mitochondrial membrane (TE Elthon, CR Stewart [1981] Plant Physiol 67: 780-784). This investigation reports the first solubilization of the L-proline dehydrogenase (PDH) from plant mitochondria. The supernatant from NP-40-treated etiolated shoot mitochondria of maize, Zea mays L., reduced iodonitrotetrazolium violet in a proline dependent manner. The pH optimum for this activity was 8. The apparent K_m for proline was 6.6 millimolar. When supplied with proline, this solubilized PDH activity also synthesized pyrroline-5-carboxylic acid. The PDH activity was inhibited in vitro by 300 millimolar potassium chloride but not by 300 millimolar potassium acetate. The PDH activity had a molecular mass that was greater than 150 kilodaltons. Mitochondria were prepared from etiolated shoots grown in 100% water-saturated vermiculite (control) and 16% water-saturated vermiculite (stress). The specific activity of solubilized PDH from the stress treatment was 11% of the same activity from the control treatment. Oxygen uptake in the presence of proline and ADP (state 3 proline oxidation) by mitochondria from the stress treatment was 25% of the same rate by mitochondria from the control treatment. Mitochondria were also prepared 16 hours after rewatering the seedlings growing in the stress treatment. Both the solubilized PDH specific activity and state 3 proline oxidation returned to the control levels. The specific activities of the NAD⁺-dependent pyrroline-5-carboxylic acid dehydrogenase and cytochrome c oxidase in the solubilized preparations were unaffected by these stress and recovery treatments. Oxygen uptake rates by intact mitochondria in the presence of ADP and NADH, succinate or malate-pyruvate were also unaffected by these treatments.     

Translocation of Some Anions, Cations, and Acids in Turnip (Brassica napus L.) Mitochondria

The translocation of various anions, cations, and acids acrossthe membrane system of turnip mitchondria has been investigatedby light scattering techniques. The results imply that the membranesof turnip mitochondria are not permeable to choline, K⁺, Na⁺,, , , and . It has been established that Cl^–,, and SCN– passively permeate the mitochondrial membrane. It is probable that permeates as NH₃ and that and cross the membrane either as the corresponding acid or on ananion/ hydroxide antiporter. The alkali metals Na⁺ and K⁺ equilibrateacross the membrane via specific H⁺/Na⁺ and H⁺/K⁺ antiporters,having similar activity at neutral pH.