Microsporogenesis and meiotic behavior in nine species of the genus Pinus

The meiotic behavior of 10 taxa （nine species and one variety） of the genus Pinus was investigated using pollen mother cells （PMCs） to reveal the differentiation among karyotypes. Chromosome spreads were prepared by conventional squashing. The meiotic index and the average configuration were higher, whereas the frequency of aberrance （chromosomal bridges, fragments, or micronuclei） was lower, in all 10 taxa compared with other gynmosperms. The meiotic index, average configuration, and frequency of irregularity were found to be uniform among the species. It was shown that the genomes of the Pinus species investigated were highly stable, confirming results of previous mitotic analyses in this genus. However, slight differentiation of homologous chromosomes among genomes was revealed by analysis of meiotic configurations in Pinus nigra var. poiretiana. Quadrivalents were observed in 9.31% of PMCs in this species. This is the first time that quadrivalents have been observed in gymnosperms.     

马尾松Ls-rDNA 5'末端序列分析及其系统学意义

马尾松Ls-rDNA 5‘末端302个核苷酸序列已被确定,与4种裸子植物和4种被子植物及一种绿藻的同源序列进行比较分析,其所构建的Ls-rDNA系统树图表明,传统分类中的裸子植物与被子植物明显成为单系类群,支持裸子植物的两个单系谱支,即苏铁目—银杏目,买麻藤目—松柏类。Ls-rDNA 5’末端部分序列分析在种子植物高等级分类群系统进化研究中具有重要作用。     

Endoplasmic reticulum preprophase band in dividing root-tip cells of Pinus brutia

In dividing root-tip cells of Pinus brutia Ten., immunolocalization of the luminal endoplasmic reticulum (ER) proteins, which have the C-terminal HDEL sequence, reveals that the ER is reorganized during the preprophase/prophase stage. Portions of ER were arrayed into a ring-like structure at the site of the microtubule preprophase band (Mt-PPB). This preprophase ER band (ER-PPB) resembles that of the Mt-PPB. The former undergoes a maturation process closely similar to that of the latter. Our data show that the PPB region has a more complex organization than is currently believed. The probable function(s) of the ER-PPB is discussed.     

Different karyotype patterns among allopatric Pinus nigra (Pinaceae) populations revealed by molecular cytogenetics

To examine variation and taxonomic recognition of Pinus nigra (European black pine) at the intraspecific level, chromosomal distribution of 5S and 18S-5.8S-26S rDNA loci revealed by fluorescent in situ hybridisation (FISH) and fluorochrome banding with chromomycin A(3) and DAPI were analysed among allopatric populations belonging to different subspecies. Despite prevalent opinion on predominantly conserved and homogenous conifer karyotypes, several patterns were observed. Surprisingly, interstitial 18S rDNA loci and DAPI heterochromatin staining after FISH showed variations in distribution and localisation. Three subspecies shared a pattern with nine 18S rDNA loci (ssp. nigra, pallasiana and laricio) while ssp. dalmatica and salzmannii had eight rDNA loci. DAPI banding displayed two patterns, one with a high number of signals (ssp. nigra, pallasiana and dalmatica) and the other with a lower number of signals (ssp. salzmannii and laricio). We conclude that our results cannot provide proof for either classification scheme for the P. nigra complex, but rather demonstrate the variability of different heterochromatin fractions at the intraspecific level.     

A novel role in cytokinesis reveals a housekeeping function for the unfolded protein response

The unfolded protein response (UPR) pathway helps cells cope with endoplasmic reticulum (ER) stress by activating genes that increase the ER's functional capabilities. We have identified a novel role for the UPR pathway in facilitating budding yeast cytokinesis. Although other cell cycle events are unaffected by conditions that disrupt ER function, cytokinesis is sensitive to these conditions. Moreover, efficient cytokinesis requires the UPR pathway even during unstressed growth conditions. UPR-deficient cells are defective in cytokinesis, and cytokinesis mutants activate the UPR. The UPR likely achieves its role in cytokinesis by sensing small changes in ER load and making according changes in ER capacity. We propose that cytokinesis is one of many cellular events that require a subtle increase in ER function and that the UPR pathway has a previously uncharacterized housekeeping role in maintaining ER plasticity during normal cell growth.     

Endoplasmic reticulum targeted GFP reveals ER organization in tobacco NT-1 cells during cell division.

The endoplasmic reticulum (ER) of plant cells undergoes a drastic reorganization during cell division. In tobacco NT-1 cells that stably express a GFP construct targeted to the ER, we have mapped the reorganization of ER that occurs during mitosis and cytokinesis with confocal laser scanning microscopy. During division, the ER and nuclear envelope do not vesiculate. Instead, tubules of ER accumulate around the chromosomes after the nuclear envelope breaks down, with these tubules aligning parallel to the microtubules of the mitotic spindle. In cytokinesis, the phragmoplast is particularly rich in ER, and the transnuclear channels and invaginations present in many interphase cells appear to develop from ER tubules trapped in the developing phragmoplast. Drug studies, using oryzalin and latrunculin to disrupt the microtubules and actin microfilaments, respectively, demonstrate that during division, the arrangement of ER is controlled by microtubules and not by actin, which is the reverse of the situation in interphase cells.     

Augmin plays a critical role in organizing the spindle and phragmoplast microtubule arrays in Arabidopsis

In higher plant cells, microtubules (MTs) are nucleated and organized in a centrosome-independent manner. It is unclear whether augmin-dependent mechanisms underlie spindle MT organization in plant cells as they do in animal cells. When AUGMIN subunit3 (AUG3), which encodes a homolog of animal dim γ-tubulin 3/human augmin-like complex, subunit 3, was disrupted in Arabidopsis thaliana, gametogenesis frequently failed due to defects in cell division. Compared with the control microspores, which formed bipolar spindles at the cell periphery, the mutant cells often formed peripheral half spindles that only attached to condensed chromosomes or formed elongated spindles with unfocused interior poles. In addition, defective cells exhibited disorganized phragmoplast MT arrays, which caused aborted cytokinesis. The resulting pollen grains were either shrunken or contained two nuclei in an undivided cytoplasm. AUG3 was localized along MTs in the spindle and phragmoplast, and its signal was pronounced in anaphase spindle poles. An AUG3-green fluorescent protein fusion exhibited a dynamic distribution pattern, similar to that of the γ-tubulin complex protein2. When AUG3 was enriched from seedlings by affinity chromatography, AUG1 was detected by immunoblotting, suggesting an augmin-like complex was present in vivo. We conclude that augmin plays a critical role in MT organization during plant cell division.     

Comparative immunofluorescence and ultrastructural analysis of microtubule organization in Uronema sp., Klebsormidium flaccidum, K. subtilissimum, Stichococcus bacillaris and S. chloranthus (Chlorophyta)

A detailed comparative examination of microtubule (MT) organization in interphase and dividing cells of Uronema sp., Klebsormidium flaccidum, K. subtilissimum, Stichococcus bacillaris and S. chloranthus was made using tubulin immunofluorescence and transmission electron microscopy (TEM). During interphase all the species bear a well-organized cortical MT system, consisting of parallel bundles with different orientations. In Uronema sp. the cortical MT bundles are longitudinally oriented, whereas in the other species they are in transverse orientation to the axis of the cells. Considerable differences in MT organization were also observed during stages of mitosis, mainly preprophase, as well as cytokinesis. In Uronema sp., a particular radial MT assembly is organized during preprophase-early prophase, which was not observed in the other species. In Stichococcus a fine MT ring surrounded the nucleus during preprophase and prophase. An MT ring, together with single cytoplasmic MTs, was also found associated with the developing diaphragm during cytokinesis in Stichococcus. A phycoplast participates in cytokinesis in Uronema sp., but not in the other species. In Uronema sp. the centrosome functions as a microtubule organizing center (MTOC) during mitosis, but not during interphase and cytokinesis. The phylogenetic significance of these differences is discussed in combination with SSU/ITS sequencing and other, existing molecular data.     

Properties of the lipid body lipase of Pinus eduUs and electrophoretic purification of its 64 kDa subunit

Lipase (triacyiglycerol acylhydrolase, EC 3.1.1.3) in oilseeds can be associated with either the lipid body or glyoxysomal membrane and can have various pH optima and substrate specificities. There is conflicting evidence for the subcellular location of lipase in gymnosperms, and little information exists on its activity characteristics. In this report, Pinus edulis (pinyon) was found to have an acid lipase, which was associated with the lipid body fraction and the activity of which increased during germination. Active lipase from the solubilized lipid body membrane was determined by gel permeation chromatography to have a molecular weight of 260 000. Further attempts to purify the active enzyme were unsuccessful. A lipid body membrane protein of 64 kDa which increased in parallel with lipase activity during germination was isolated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. excised, and polyclonal antibodies were made against it. Using these antibodies, active lipase was immunoprecipitated from solution, thus indicating that the 64 kDa protein is a subunit of the lipase. Pinus edulis lipase had a pH optimum of ca 4.5. and it exhibited little specificity for triacyiglycerol substrates in vitro. The lipase was specific in activity against fluorometric substrates, with the highest activity against methyl-umbelliferyl laurale. Lipase activity was inhibited by high concentrations of non-ionic detergent. This lipid body acid lipase appears to be primarily responsible for lipid hydrolysis during pinyon germination.     

Enzymatic Isolation of Archegonium of Pinus Yunnanensis Fr. and Observations on the Proembryo Development

The enzymatic isolation of megagametophyte from fixed and fresh of Pinus yunnanensis Fr. were macerated in a mixture solution of 3% pectinase and 3% cellulase at the 30℃ for 10–13 hours, and the archegonium has been isolated. Materials were stained with A-S, Feulgen and PAS, and following procedure was cleared in the lactophenol. The ventral canal and egg cell which from division of central cell, the functional sperm nucleus becomes pressed against the egg nucleus, and the new cytoplasm round the zygote nucleus were observated under the microscope of usual light. The four proembryonal nuclei divide simultaneously, resulting in eight nuclei arranged in two tiers of four cells each, and following development were formed wall. In the end, the proembryo of six- teen cells were formed. Two methods on enzymatic and dimethyl sulfoxide with potassium hydroxide to isolate the female sexual cells of gymnosperms have been compared, and several problems are discussed.     

Mycorrhizal associations in Pinus massoniana Lamb. and Pinus elliottii Engel. inoculated with Pisolithus tinctorius

Dichotomous mycorrhizas were induced in Pinus massoniana and Pinus elliottii seedlings inoculated with Pisolithus tinctorius growing under non-axenic conditions. Six months after inoculation, Pinus massoniana seedlings exhibited a higher degree of infection, bore more mycorrhizas and had developed more abundant extramatrical mycelium than seedlings of Pinus elliottii. Nevertheless, seedlings of Pinus massoniana were stunted and exhibited chorosis of the needles, indicating a possible nutrient deficiency. Histological examination of these pine mycorrhizas showed an ectomycorrhizal association typical of gymnosperms with an intercellular Harting net penetrating between several layers of cortical cells close to the endodermis. However, strong polyphenolic reactions, intracellular hyphae and wall modifications were occasionally observed, indicating that both host-tissue incompatibility and ectendomycorrhizal association can occur in pine species under stressed conditions.     

A Novel Plant Kinesin-Related Protein Specifically Associates with the Phragmoplast Organelles

In higher plants, the formation of the cell plate during cytokinesis requires coordinated microtubule (MT) reorganization and vesicle transport in the phragmoplast. MT-based kinesin motors are important players in both processes. To understand the mechanisms underlying plant cytokinesis, we have identified AtPAKRP2 (for Arabidopsis thaliana phragmoplast-associated kinesin-related protein 2). AtPAKRP2 is an ungrouped N-terminal motor kinesin. It first appeared in a punctate pattern among interzonal MTs during late anaphase. When the phragmoplast MT array appeared in a mirror pair, AtPAKRP2 became more concentrated near the division site, and additional signal could be detected elsewhere in the phragmoplast. In contrast, the previously identified AtPAKRP1 protein is associated specifically with bundles of MTs in the phragmoplast at or near their plus ends. Localization of the tobacco homolog(s) of AtPAKRP2 was altered by treatment of brefeldin A in BY-2 cells. We discuss the possibility that AtPAKRP1 plays a role in establishing and/or maintaining the phragmoplast MT array, and AtPAKRP2 may contribute to the transport of Golgi-derived vesicles in the phragmoplast.     

Chromosomal localization of 5S and 18S rDNA in five species of subgenus Strobus and their implications for genome evolution of Pinus

BACKGROUND AND AIMS: Studying the genome structure of pines has been hindered by their large genomes and uniform karyotypes. Consequently our understanding of the genome organization and evolutionary changes in different groups of pines is extremely limited. However, techniques are now available that can surmount these difficulties. The purpose of this study was to exploit some of these techniques to characterize the genome differentiation between the two subgenera of Pinus: Pinus and Strobus. METHODS: Double-probe fluorescence in-situ hybridization (FISH) was used to localize the 5S and 18S rDNA loci on chromosomes of five species from the subgenus Strobus: P. bungeana, P. koraiensis, P. armandii, P. wallichiana and P. strobus. * KEY RESULTS: The rDNA FISH pattern varied considerably among the five species, with P. bungeana being the most distinct. By comparing the results obtained with those of previous rDNA FISH studies of members of the subgenus Pinus, several general features of rDNA loci distribution in the genus Pinus can be discerned: (a) species of subgenus Strobus generally have more rDNA loci than species of subgenus Pinus, correlating with their larger genomes in the subgenus Strobus; (b) there is a clear differentiation in 5S and 18S rDNA loci linkage patterns between the two subgenera; (c) variations in the rDNA FISH pattern correlate with phylogenetic relationships among species within the subgenus; (d) P. bungeana has fewer 18S rDNA sites than other pines investigated to date, but they give intense signals, and may reflect the primary distribution of the 18S-25S rDNA loci in the genus. CONCLUSIONS: The stable differentiation in rDNA FISH pattern between the subgenera suggests that chromosomal rearrangements played a role in the splitting of the two subgenera, and transpositional events rather than major structural changes are likely responsible for the variable rDNA distribution patterns among species of the same subgenus with conserved karyotypes.     

Late Embryogeny of Pinus bungeana

Except for Pseudolarix, which is endemic to China, the late embryogeny of ten genera of Pinaceae has been reported before in the diffferent degree. Among them the mature embryos of Keteleeria evelyniana are different from those of the others in having well-developed cotyledons and a very short hypocotyl. As far as information we have is concerned there are three types in structure of the mature embryos of Pinaceae. The first type occurs in Keteleeria and Cedrus, which have very well-developed cotyledons; the second one appears in Picea and Larix, in which the hypocotyl and the root cap are equal in length; the third one, to which Pinus bungeana belongs, includes all the other genera of Pinaceae. The last type is of a prominent hypocotyl in the mature embryos. It is interesting to note that the mature embryos of Torreya grandis and Keteleeria evelyniana are very similar in having specially developed cotyledons, while the proportions of the various tissues in the mature embryo in Fokienia of Cupressaceae as well as Taxus and Amentotaxus of Taxaceae are similar to those of Pinus bungeana. The pith and secretory cells are usually present in the mature embryos of Pinaceae. Although no pith is present in those of Metasequoia and Taiwania of Taxodiaceae, the secretory cells generally occur in their embryos in Fokienia of Cupressaceae and Taxus and Pseudotaxus of Taxaceae, neither pith nor secretory cells are present in their hypocotyl. From above, the structures of mature embryos among Pinaceae, Taxodiaceae and Cupressaceae are different from one another to some degree. The most outstanding feature of the matur embryos in Pinus bungeana is that the shoot apex is very well developed, with a high H/D ratio, about 0.83 on an average, even up to 0.96 in some case. Above-mentioned H/D ratio of Pinus bungeana is rare in the mature embryos of conifers. Gifford (1943) reports that the average H/D ratio of shoot apex of Ephedra altissima is from 0.44 to 0.68, while in 5-year-old branch apex of Pinus ponderosa, the average ratio is about 0.25, and that in l5-year-old branch apex is about 0.35. For the apex of the dormant short shoots of Pinus densiflora the average ratio is about 0.35, but that of shoot apex is about 0.52 when new buds have just formed. According to the present data about shoot apices of both branches and mature embryos, the average ratio of shoot apex of mature embryos in Pinus bungeana is the largest one. From the present investigation the shoot apex of mature embryos of Pinus bungeana exhibits four distinct tissue zones, i.e. the apical initials, the central mother cell zone, the peripheral tissue zone and the rib meristem. It is worthy of note that the shoot apex of Pinus strobus may be divided into five zones, including transition zone between central mother cells and rib meristem (Owston, 1968). Four zones are recognized in the shoot apex of Pinus lambertiana and P. ponderosa, without transition one (Sacher, 1954). From cytological zonation, the shoot apex of mature embryos in Pinus bungeana is rather similar to that of Pinuslambertiana and P. ponderosa.     

Visualization of actin cytoskeletal dynamics during the cell cycle in tobacco (Nicotiana tabacum L. cv Bright Yellow) cells

BACKGROUND INFORMATION: The actin cytoskeleton forms distinct actin arrays which fulfil their functions during cell cycle progression. Reorganization of the actin cytoskeleton occurs during transition from one actin array to another. Although actin arrays have been well described during cell cycle progression, the dynamic organization of the actin cytoskeleton during actin array transition remains to be dissected. RESULTS: In the present study, a GFP (green fluorescent protein)-mTalin (mouse talin) fusion gene was introduced into suspension-cultured tobacco BY-2 (Nicotiana tabacum L. cv Bright Yellow) cells by a calli-cocultivation transformation method to visualize the reorganization of the actin cytoskeleton in vivo during the progression of the cell cycle. Typical actin structures were indicated by GFP-mTalin, such as the pre-prophase actin band, mitotic spindle actin filament cage and phragmoplast actin arrays. In addition, dynamic organization of actin filaments was observed during the progression of the cell from metaphase to anaphase. In late metaphase, spindle actin filaments gradually shrank to the equatorial plane along both the long and short axes. Soon after the separation of sister chromosomes, actin filaments aligned in parallel at the cell division plane, forming a cylinder-like structure. During the formation of the cell plate, one cylinder-like structure changed into two cylinder-like structures: the typical actin arrays of the phragmoplast. However, the two actin arrays remained overlapping at the margin of the centrally growing cell plate, forming an actin wreath. When the cell plate matured further, an actin filament network attached to the cell plate was formed. CONCLUSIONS: Our results clearly describe the dynamic organization of the actin cytoskeleton during mitosis and cytokinesis of a plant cell. This demonstrates that GFP-mTalin-transformed tobacco BY-2 cells are a valuable tool to study actin cytoskeleton functions in the plant cell cycle.     

裸子植物5S rRNA基因序列变异及二级结构特征

在高等植物中,5SrRNA基因一级结构是高度保守的,二级结构也相当一致。通过比较18种裸子植物5SrRNA基因序列和二级结构变异,发现55％的核苷酸位点是可变的,这种变异有68％发生在干区（双链区）,其中一些变异,如双链的互补性核苷酸替代,GU配对等能够维系5SrRNA二级结构的稳定性。环区相对保守,这与5SrRNA三级结构折叠或在转录翻译过程中蛋白质、RNA的结合相关。另外,首次报道了松属环E区核苷酸的变异性,这可能与其他区域的变异一样,是假基因造成的结果。5SrRNA基因信息可反映大分类群的系统进化关系,但由于基因长度短,信息量小,其在近缘种系统分类的应用受到限制。     

Organization of microtubules in dividing and elongating cells of Vicia hajastana Grossh. in suspension culture

Cells of Vicia hajastana Grossh. cultured with 2,4-D showed coupled division and growth and formed multicellular files of small isodiametric cells. In GA without added 2,4-D, the cells stopped dividing and continued elongating for several days. Total growth was the same in both hormone conditions. An immunofluorescent technique was developed to study microtubule (MT) distribution. Cells in GA showed parallel MT arrays oriented transversely to the axis of elongation. In some cells the number of MT per unit length was maintained during growth while other elongating cells showed reduced frequency of MT. Microtubules often appeared as thickened, branched strands, probably as a result of lateral aggregation. In cells grown in 2,4-D some pre-prophase bands of MT were observed. Cells in mitosis lacked cortical MT, and all organized staining was in spindles or phragmoplasts. Interphase cells in 2,4-D showed variable organization of cortical MT ranging from disordered to transversely ordered. Cells in early interphase had disordered MT while larger cells showed order. These observations indicate that MT in cycling cells are continually changing organization, probably accounting for the different distributions observed in interphase cells. On cessation of the mitotic cycle, reorganization of MT stops and transverse arrays of cortical MT are maintained as cells elongate. These processes are similar to those observed in organized tissues; however, cultured cells offer distinct advantages for experimental manipulation and microscopic observation of cytoskeleton.     

Structural organization and dynamics of the endoplasmic reticulum during spermatogenesis of Drosophila melanogaster: Studies using PDI-GFP chimera protein

An important property of the endoplasmic reticulum (ER) is its ability to change morphology and intracellular localization during the cell cycle and differentiation. Visualization of the ER membranes using the protein disulphide isomerase (PDI) GFP chimeric protein makes it possible to trace the dynamics of all these processes and expose transitional forms and intermediate configurations. In this article the results of the study of the ER morphology during spermatogenesis of D. melanogaster are presented. It was shown that ER membranes retain high level of GFP-fluorescence through all stages of spermatogenesis, so that revealing of the stage-specific features of the ER organization was possible. The ER network has distinctive reticular morphology during the interphase and early prophase. Right before the cell division, this morphology changes and ER forms a system of branchless filamentous membranes. In prometaphase, these membranes form concentric circles adjacent to the nuclear membrane; from metaphase to telophase, they lengthen along the axis of cell division and resemble a spindle. Later, in the next interphase, this configuration transforms to a reticular structure seen previously. At the beginning of spermatid differentiation, the ER encompasses nebenkern and nucleus and elongates adjacent to them. During the latest stages of spermatogenesis, the ER network dissociates into separate membranous granules that are eliminated from the cyst with the individualization complex. Possible mechanisms of the ER dynamics and reorganization are discussed.     

The contribution of short repeats of low sequence complexity to large conifer genomes

The abundance and genomic organization of six simple sequence repeats, consisting of di-, tri-, and tetranucleotide sequence motifs, and a minisatellite repeat have been analyzed in different gymnosperms by Southern hybridization. Within the gymnosperm genomes investigated, the abundance and genomic organization of micro- and minisatellite repeats largely follows taxonomic groupings. We found that only particular simple sequence repeat motifs are amplified in gymnosperm genomes, while others such as (CAC)₅ and (GACA)₄ are present in only low copy numbers. The variation in abundance of simple sequence motifs reflects a similar situation to that found in angiosperms. Species of the two- and three-needle pine section Pinus are relatively conserved and can be distinguished from Pinus strobus which belongs to the five-needle pine section Strobus. The hybridization pattern of Picea species, bald cypress and gingko were different from the patterns detected in the Pinus species. Furthermore, sequences with homology to the plant telomeric repeat (TTTAGGG)_n have been analyzed in the same set of gymnosperms. Telomere-like repeats are highly amplified within two- and three- needle pine genomes, such as slash pine (Pinus elliottii Engelm. var. elliottii), compared to P. strobus, Picea species, bald cypress and gingko. P. elliottii var. elliottii was used as a representative species to investigate the chromosomal organization of telomere-like sequences by fluorescence in situ hybridization (FISH). The telomere-like sequences are not restricted to the ends of chromosomes; they form large intercalary and pericentric blocks showing that they are a repeated component of the slash pine genome.Conifers have genomes larger than 20000 Mbp, and our results clearly demonstrate that repeats of low sequence complexity, such to (CA)₈, (GA)₈, (GGAT)₄ and (GATA)₄, and minisatellite- and telomere-like sequences represent a large fraction of the repetitive DNA of these species. The striking differences in abundance and genome organization of the various repeat motifs suggest that these repetitive sequences evolved differently in the gymnosperm genomes investigated. Received: 1 October 1999 / Accepted: 3 November 1999