RNA Synthesis and polysome formation in pollen tubes

The formation of polysomes in relation to RNA synthesis was investigated in tobacco (Nicotiana tabacum L.) pollen cultivated submersely for a period of 12 h. The percentage of polysomes was estimated by determining the number of ribosomes carrying nascent polypeptides using RNase and 0.8 M KC1 treatment of the ribosomal preparation. This approach showed that the proportion of ribosomes “active” in protein synthesis amounts to about 12 % in dry pollen rising to 46 % within 10 min of imbibition and to 66 % during the period 1–4 h of cultivation. The latter increase is accompanied by a rapid incorporation of uridine-¹⁴C into polysome-as-sociated RNA and is sensitive to actinomycin D. The rapidly labelled RNA isolated from the ribosomal preparation sedirnented in the range from about 5S to 30S. Longer labelling periods led to a gradual shift of the major peak of this heterogeneous RNA from about 11.5S and 14S to about 7.5S and the development of radioactivity peaks in the position of 18S and 28S RNA. Uridine incorporated both into the active ribosomes and into the subunits of inactive ribosomes at a more or less constant rate during the whole 12-h period of cultivation. These results present evidence that in addition to the initial combination of the existing ribosomes with the stored mRNA following imbibition, an activation of pollen tube genome and its implication in directing protein synthesis take place during the early phases of pollen tube growth. From the results on kinetics of labelling of ribosomes it appears that in pollen tubes new synthesized ribosomal subunits enter polysomes directly, the entry of 40S subunits being more rapid than that of 60S subunits.     

On the nature of RNA synthesized in pollen tubes ofNicotiana alata

The RNA formed in pollen tubes during 4 hours of growthin vitro was resolved by chromatography on methylated albumine on kieselguhr (MAK) into three principal fractions. Acoording to the labelling from uracil-¹⁴C about 11% was eluted with tRNA and 5 S RNA (low molecular weight RNA), 76% just after rRNA (D-RNA) and nearly 14% was recovered from the column by SDS at 35 °C (TB-RNA). In the presence of actinomycin D at concentration of 30 μg ml^-1 the synthesis of the three classes of RNA was inhibited by 71%, 97% and 70% respectively. On sucrose density gradient the radioactive low molecular weight RNA sedimented at 4 S-5 S which suggests that one or both of these RNA species are synthesized in pollen tubes. The D-RNA eluted from the MAK column is polydisperse in size exhibiting a wide range of sedimentation values up to about 35 S with a large peak at 9 S-10 S and two smaller peaks at 14 S-15 S and at about 23 S. The rapid labelling and the polydisperse rather low molecular weight character suggest that the D-RNA is a heterogeneous population of mRNA. The sedimentation profile of TB-RNA was similar to that of D-RNA. The RNA synthesized in the presence of³²PBO3-₄ or uracil-¹⁴C exhibited no radioactivity peaks corresponding to sedimentation peaks of rRNA.     

Chromosome location of the genes for 28S, 18S and 5S ribosomal RNA in Triturus marmoratus (Amphibia Urodela)

The localization of the 28S, 18S and 5S rRNA genes in the mitotic chromosomes, and of the 5S rRNA genes in the lampbrush chromosomes of Triturus marmoratus has been studied by RNA/DNA in situ hybridization. The 28S and 18S genes are located in a subterminal position, and the 5S genes in an intermediate position, on the long arm of mitotic chromosome X. In situ hybridization on lampbrush chromosomes has shown that the 5S genes are located at or near a dense matrix loop landmark. The cytogenetic implications of these findings are briefly discussed.     

The effect of 2-thiouracil on RNA synthesis in pollen tubesof Nicotiana alata

The level of RNA in pollen is approximately 20 mg g^-1 and remains constant during 6 h pollen germinationin vitro also in the presence of 2-thiouracil which stimulates pollen tube elongation. The synthesis of RNA in pollen tubes was investigated according to the incorporation of the label from uracil-2-¹⁴C, 2-thiouracil-2-¹⁴C, orotic acid-5-³H, fructose-U-¹⁴C and from³²PO₄ ^3- into RNA fractions separated by methylated albumine kieselguhr chromatography. The distribution of radioactivity on elution profiles was different according to the radioactivity source, however it was not changed by the presence of 2-thiouracil in cultivation medium. 2-Thiouracil incorporates into pollen tube RNA at about 50% the rate of uracil. It inhibited the incorporation of orotic acid, of fructose and of phosphate into all RNA fractions. It is suggested that the analogue inhibits the enzymes involved in RNA synthesis essentially as 2-thiouridine-5’-phosphate.     

Nucleotide sequence of 5S RNA ofMycobacterium smegmatis

³²P labelled 5S RNA isolated fromMycobacterium smegmatis was digested withT ₁ and pancreatic ribonucleases separately and fingerprinted by two dimensional high voltage electrophoresis on thin-layer DEAE-cellulose plates. The radioactive spots were sequenced and their molar yields were determined. The chain length of the 5S RNA was found to be 120. It showed resemblances to both prokaryotic and eukaryotic 5S RNAs.     

Variation in organisation and copy number of ribosomal RNA genes inPetunia hybrida somaclones

The copy number of genes encoding 5S ribosomal RNA has been found to be constant in Petunia hybrida plants regenerated from protoplast and leaf disc-derived callus cultures. However, in one somaclone a heritable change in the length of the major 5S rDNA repeat has arisen. Despite the constant copy number of 5S rRNA genes, that of the 18S–25S rRNA genes is found to very by at least ten-fold. The relevance of these findings to ribosomal RNA gene variability and to somaclonal variation is discussed.     

Microsporogenesis,pollen mitosis and pollen tube growth in Gagea villosa (Liliaceae)

In this study, Gagea villosa (Bieb.) Duby was investigated by using light microscopy methods in cytological and cytoembryological respects. Anthers were tetrasporangiate. Anther wall was formed with an epidermis, endothecium, middle layer and tapetum. Tapetum was glandular type and it began to degenerate when microspores released from tetrads. Tapetum cells generally have one or two nuclei. Mitosis seen in tapetum cells was generally normal but micronuclei were found in some of them. Fibrous thickenings were determined in endothecium. Microsporogenesis and pollen mitosis were generally regular. Cytokinesis was successive type. Meiosis in pollen mother cells was asynchronous in one anther locus. Mature pollen grains were 2-celled. Pollen sterility was found to be 24%. Some of the fertile pollen grains, smaller than the normal were seen at the end of the pollen mitosis. Microgametophyte development was examined in vivo and in vitro. Germination ratio of pollen grains in vitro was 4%. Generally swollen pollen tube tips and weak development of some curled pollen tubes were seen. Callose plug formation was seen only in vivo pollen tube growth.     

tRNA synthesis in germinating pollen

The synthesis of tRNA was demonstrated in pollen ofNicotiana tabacum L. according to the incorporation of labeled uracil, adenosine and guanosine during 4 h of germination. tRNA was extracted from postribosomal supernatant and purified by polyacrylamide gel electrophoresis. The incorporation of guanosine was about 1.68 times higher than that of adenosine. This finding indicates that the whole tRNA chain is formed in pollen tubes.     

Cytometric analysis of growth-regulator-dependent transcription and cell-cycle progression in Petunia protoplast cultures

Acridine orange simultaneously stains DNA and RNA. Using flow cytometry, synthesis of these nucleic acids can be related throughout a culture time-course. This technique has been used with nuclei isolated from Petunia hybrida protoplasts during 48 h of culture. Nuclear RNA content has been evaluated with respect to DNA levels, namely the cell-cycle phase. Nuclear RNA synthesis was not dependent upon exogeneous hormones during the first 18 h of culture, but either auxin (2,4-dichlorophenoxyacetic acid, 2,4-D) or cytokinin (N⁶-benzyladenine) were necessary for entry into the S phase. Cytokinin alone could stimulate maximal RNA synthesis within each cell-cycle phase up to 24 h. In complete medium, DNA synthesis only began from a phase “G₁B” having substantial RNA, although a subnormal amount of RNA (in protoplasts cultivated only with 2,4-D) did not prevent protoplast entry into the S phase. However, both hormones were necessary for highest RNA levels and G₂ frequencies after 48 h. As in mammalian cells, the mean RNA level in plant 4C nuclei is double that of 2C nuclei. G₂ nuclei are larger than G₁ nuclei, but upon activation G₁ nuclei in fact diminsh in size. This study aimed to identify restriction points in the cell cycle as affected by growth regulators and the specific synthesis of nucleic acids. For example, the RNA levels induced by N⁶-benzyladenine, although similar to those in complete medium, were not sufficient to induce mitosis. Conversely, 2,4-D action was probably limited by low nucleotide synthesis in the absence of cytokinin.     

Pistillate flower development and pollen tube growth mode during the delayed fertilization stage in Corylus heterophylla Fisch

Unlike most angiosperms, in which fertilization occurs within several days after pollination, fertilization in hazel (Corylus Spp.) is delayed by two to three and a half months. However, the female inflorescences or young fruits are too hard or lignified to be dissected according to regular paraffin sectioning technique. So, what the nature of development during the extended progamic phases of hazel remains unknown. The female inflorescence development and pollen tube growth mode during the delayed fertilization stage in hazel were investigated by improved paraffin sectioning and aniline blue staining of pollen tubes. The results showed ovaries and ovules of hazel were invisible at the time of blooming. Early ovary and ovule primordium began to form from 15 to 20 days after blooming, respectively. Integument and mature embryo sacs differentiated from the nucellus on 40th and 55th day after blooming, respectively. Pollen tubes were retarded in the bottom of the style or the pollen tube cavity (PTC, a specifical lignified cavity structure at the bottom of style for pollen tube to rest during progamic phase) for about 26 days. Then, the pollen tubes were observed to leave the PTC and began to enter the ovary. After that, a single pollen tube passed through the vicinity of the micropyle. Finally, pollen tubes turned a corner and penetrated the embryo sac through the tissue of the chalaza instead of micropyle on 52 and 55 days after blooming, respectively. The results of more in-depth information will be beneficial to better understanding of the delayed fertilization process in hazel.     

Sucrose concentration in the growth medium affects the cell wall composition of tobacco pollen tubes

The cell wall of pollen tubes is organized in both spatial and temporal order to allow the pollen tube to grow according to external conditions. The deposition of methyl-esterified and acid pectins in addition to callose/cellulose occurs according to a series of temporally succeeding events. In this work, we attempted to determine how the composition of the external growth medium (in terms of osmolarity) could affect the deposition of cell wall components. Pollen tubes of tobacco were grown in a hypotonic medium and then analyzed for the distribution of pectins and callose/cellulose [as well as for the distribution of the enzyme callose synthase (CALS)]. The data indicate that pollen tubes grown in a hypotonic medium show changes of the initial growth rate followed by modification of the deposition of acid pectins and, to a lesser extent, of CALS. These observations indicate that, under the osmolarity determined by the growth medium, pollen tubes adapt their cell wall to the changing conditions of growth.     

Synthesis of protein and RNA in pollen tubes stimulated with 2-thiouracil

1. 1.
   Protein synthesis in pollen tubes ofNicotiana alata Link etOtto estimated by the incorporation of leucine-¹⁴C is linear over six hours of artificial cultivation after a short lag phase. 2-Thiouracil and other growth-stimulating antimetabolites of natural pyrimidines and purines, such as 6-azauracil, 5-nitrouracil, 8-azaguanine and 8-azaadenine, stimulate the incorporation of leucine-¹⁴C into protein. The intensity of stimulation of protein synthesis is associated with the intensity of growth stimulation by antimetabolite.     
   
   

Alterations in polyadenylated RNA during pollen maturation and germination

Developmental changes in poly(A)-bearing RNA in male tobacco gametophyte were examined by sedimentation analysis and by hybridization with³H-poly(U). The results indicate that the transition of microspore undergoing postmeiotic division to mature pollen is accompanied by characteristic changes in RNA and poly(A) content and the size of poly(A)⁺RNA. The volume of pollen grain increases about 2times, total RNA per grain from 34 to 230 pg and poly(A) from 22 to 450 fg, which together with the estimated increase in the number average size of poly(A)⁺RNA from 700 to 2 100 nucleotides suggests an approx. rise of RNA containing poly(A) from 0.3 to 2.7% of total RNA. Size distribution of the populations of polyadenylated RNAs shows progressive formation of species with a higher molecular mass and differentiation of the pollen-characteristic pattern with main sedimentation maxima close to 12S, 19S and 26S. This pattern remains almost unchanged during 8 h of pollen tube growth and is also found in polysomes formed at the beginning of germination. The amount of poly(A) decreases gradually after the onset of soaking at a rate of slightly more than 1 % per h within 24 h of pollen cultivation. As a whole, the results demonstrate that in the course of pollen maturation a specific population of polyadenylated mRNAs is formed which persists as stored mRNA in quiescent pollen and is used as template during-pollen tube formation.