Peeking into pit fields: a multiple twinning model of secondary plasmodesmata formation in tobacco

In higher plants, plasmodesmata (PD) are major conduits for cell-cell communication. Primary PD are laid down at cytokinesis, while secondary PD arise during wall extension. During leaf development, the basal cell walls of trichomes extend radially without division, providing a convenient system for studying the origin of secondary PD. We devised a simple freeze-fracture protocol for examining large numbers of PD in surface view. In the postcytokinetic wall, simple PD were distributed randomly. As the wall extended, PD became twinned at the cell periphery. Additional secondary pores were inserted at right angles to these, giving rise to pit fields composed of several paired PD. During wall extension, the number of PD increased fivefold due to the insertion of secondary PD. Our data are consistent with a model in which a subset of the original primary PD pores function as templates for the insertion of new secondary PD, spatially fixing the position of future pit fields. Many of the new PD shared the same wall collar as the original PD pore, suggesting that new PD pores may arise by fissions of existing PD progenitors. Different models of secondary PD formation are discussed. Our data are supported by a computational model, Plasmodesmap, which accurately simulates the formation of radial pit fields during cell wall extension based on the occurrence of multiple PD twinning events in the cell wall. The model predicts PD distributions with striking resemblance to those seen on fractured wall faces.     

Immunodetection and immunolocalization of globulin storage proteins during zygotic and somatic embryo development in Zea mays

Globulins (GLB) are storage proteins that accumulate to high levels during zygotic embryo development of Zea mays L. We visualized the distribution of GLB during zygotic embryo development by immunolabelling of polyethylene glycol sections with a GLB-specific antiserum and a fluorescent secondary antibody. In sections of embryos at 10 days after pollimation (DAP), GLB were detected in the scutellar node only. Sections of embryos of 17 DAP showed, besides the presence of GLB in the scutellar node, the presence of a low amount of GLB in the coleoptile and the leaf primordia. In 30-DAP embryos GLB were localized in the root, the coleorhiza, the leaf primordia, the coleoptile and in all cells of the scutellum with the exception of the epidermis and the pro-vascular tissues. The subcellular location of GLB was visualized by immunolabelling of ultrathin sections with anti-GLB and a gold-conjugated secondary antibody. Scutellum cells and root cortex cells of 30-DAP embryos were packed with protein storage vacuoles (PSV), which differed in electron density. GLB were either evenly distributed throughout the PSV or were localized in electron-dense inclusions within the PSV. SDS-PAGE and immunoblot analysis of total protein extracts indicated the presence of a low amount of the GLB1 processing intermediate proGLB1' in globular as well as mature somatic embryos. After maturation on an ABA-containing medium, somatic embryos showed the additional presence of the next GLB1 processing intermediate GLB1'. By immuno-electron microscopy it was possible to localize GLB in globular deposits in PSV in scutellum cells of these somatic embryos.     

Immunodetection and immunolocalization of globulin storage proteins during zygotic and somatic embryo development in Zea mays

Globulins (GLB) are storage proteins that accumulate to high levels during zygotic embryo development of Zea mays L. We visualized the distribution of GLB during zygotic embryo development by immunolabelling of polyethylene glycol sections with a GLB-specific antiserum and a fluorescent secondary antibody. In sections of embryos at 10 days after pollimation (DAP), GLB were detected in the scutellar node only. Sections of embryos of 17 DAP showed, besides the presence of GLB in the scutellar node, the presence of a low amount of GLB in the coleoptile and the leaf primordia. In 30-DAP embryos GLB were localized in the root, the coleorhiza, the leaf primordia, the coleoptile and in all cells of the scutellum with the exception of the epidermis and the pro-vascular tissues. The subcellular location of GLB was visualized by immunolabelling of ultrathin sections with anti-GLB and a gold-conjugated secondary antibody. Scutellum cells and root cortex cells of 30-DAP embryos were packed with protein storage vacuoles (PSV), which differed in electron density. GLB were either evenly distributed throughout the PSV or were localized in electron-dense inclusions within the PSV. SDS-PAGE and immunoblot analysis of total protein extracts indicated the presence of a low amount of the GLB1 processing intermediate proGLB1'in globular as well as mature somatic embryos. After maturation on an ABA-containing medium, somatic embryos showed the additional presence of the next GLB1 processing intermediate GLB1. By immuno-electron microscopy it was possible to localize GLB in globular deposits in PSV in scutellum cells of these somatic embryos.     

An extraction method for tobacco mosaic virus movement protein localizing in plasmodesmata

Summary. The intercellular communication by plasmodesmata (PD) is important for the growth and development of plants, and the transport of macromolecules through PD is likely to be regulated by developmental signals. While PD in the apical meristem transport macromolecules such as mRNAs, the branched PD in the mature leaf do not transport large macromolecules freely. The changes in PD during development might be important for sink-to-source changes in leaves, but the molecular mechanism is still unknown. Movement proteins (MPs) of the tobacco mosaic virus localize in the branched PD and increase the size exclusion limit, allowing transport of viral RNA. We developed a method for differential extraction of MP from isolated cell walls of transgenic tobacco leaves expressing MP or MP tagged with green-fluorescent protein. Lithium chloride at a concentration of 8 M removed filamentous structures in branched PD, the possible attachment site of MP. As some endogenous proteins were coeluted with MP by the treatment, this extraction method might be a powerful tool for investigating MP-interacting proteins in branched PD. Correspondence and reprints: Department of Life Sciences, Graduate School of Arts and Sciences, University of Tokyo, Komaba 3-8-1, Meguro-ku, Tokyo 153-8902, Japan.     

Scanning Electron Microscopic Observation of Symbiotic Relationship of Azolla-Anabaena AzoUae During the Vegetative Growth

The occurrence and development of the hair ceils on the shoot tips and in the leaf cavities of A. filiculoides, A. microphylla, A. pinnata and their algae-free cultures were examined by means of scanning electron microscopy with microdissect technique. The patterns of Anabacna moving into the leave cavities from the shoot tips were investigated on three species of Azolla during their vegetative growth. The results showed that the patterns of symbiotic Anabaena infecting the leaf cavities are similarity among three species of Azolla and may be divided to the four phases which are summarized as follows: 1. occurrence of primary branched hair and adhesion of Anabaena; 2. development of primary branched hair and spreding of Anabaena; 3. building of hair bridge and entrance of Anabaena into the cavities; 4. formation of secondary simple hair and transference of Anabaena within the cavity. These observations resulted in a hypothesis that hair induces and leads its partner. It is suggested that the hair cell is likely to be a structure of Azolla for attracting and recognizing its symbiont in addition to transport substance between fern and algae.     

Regulative interaction of mature imaginal disc Fragments with embryonic and immature disc tissues inDrosophila

Summary These experiments examined whether inDrosophila immature imaginal disc tissue and tissues from embryonic stages can influence pattern regulation in a disc fragment in the same way as can mature imaginal discs. Immature imaginal discs, or the cells of whole embryos, were mixed with a test fragment (presumptive notum) from a mature wing disc. The immature tissues in each mixture were genetically marked and had been heavily irradiated (25 Kr gamma) prior to mixing to prevent growth and maturation during subsequent culture in vivo. Alteration of the regulative behavior of the test fragment (that is, regeneration of wing) thus provided an assay for the communication of positional information by the immature tissues. The results suggest that this capacity arises well before competence to metamorphose, as early as the 16th hour of embryonic development, whereas prior to 16 h, essentially no stimulation of regeneration occurred. It is suggested that the imaginal disc (or presumptive disc) cells of the embryo may have been responsible for this early stimulatory capacity.     

Histological Observations on Initiation and Morphogenesis in Immature and Mature Embryo Derived Callus of Barley (Hordeum vulgare L.)

Callus was induced from immature and mature embryos of barley(cv. Haruna Nijo) on Murashige and Skoog medium containing 2mg l^-1 2,4-D and 5 mg l^-1 picloram, respectively. Paraffin sections(10 µm thick) were prepared for histology during callusinitiation and plant regeneration. Meristems were regeneratedfrom nodular compact callus (NC) derived from scutellar epidermisin immature embryos, whereas they were regenerated from NC derivedfrom epidermal cells of leaf or coleoptile bases in mature embryos.Regardless of the explant source, regeneration was predominantlythrough organogenesis, although regeneration through somaticembryogenesis infrequently occurred. Thus, the callus inducedfrom immature and mature embryos of barley was regarded as 'nodularcompact' rather than 'embryogenic'.Copyright 1995, 1999 AcademicPress Barley, callus, Hordeum vulgare, histology, immature embryo, mature embryo, regeneration     

Directional cell-to-cell communication in theArabidopsis root apical meristem II. Dynamics of plasmodesmatal formation

Summary Cell development in the root apical meristem is thought to be regulated by position-dependent information, but as yet, the underlying mechanism for this remains unknown. In order to examine the potential involvement of the symplasmic transmission of positional signals, plasmodesmatal frequency and distribution was quantitatively analyzed in root apical meristem cell walls ofArabidopsis thaliana during root development. A consistent distribution pattern of plasmodesmata was observed in the root apex over four weeks. While cells within initial tiers were uniformly interconnected, more symplasmic connections between the initial tiers and their immature-cell (primary-meristem) derivatives were observed than within the initial tiers. Immature cells were connected across transverse walls by primary plasmodesmata according to a tissue-specific pattern. Cells of the immature vascular tissue and cortex had the highest plasmodesmatal frequencies, followed by the immature epidermis and root cap. Although the numbers of plasmodesmata in transverse walls (primary plasmodesmata) was reduced in all tissues as the root aged, the tissue-specific distribution remained constant. The extent of symplasmic coupling across the boundaries of each tissue appeared to be limited by fewer secondary plasmodesmata in longitudinal walls. The frequency of all plasmodesmata decreased as the root aged. The primary plasmodesmata within each tissue increased at one week and then dramatically decreased with root age; the frequency of secondary plasmodesmata in longitudinal walls also decreased, but more gradually. These findings are discussed with respect to the roles likely played by plasmodesmata in facilitating transport of position-dependent information during root development.     

Periodicity in the development of the root system of young rubber trees (Hevea brasiliensis Müell. Arg.): relationship with shoot development

The growth pattern of the root system of young rubber trees (Hevea brasiliensis) was studied in relation to shoot development over a period of 3 months. Temporal and spatial variations in elongation and branching processes were examined for the different root types, by means of root observation boxes. Shoot growth was typically rhythmic. Root development was periodic and related to leaf expansion. Root elongation was depressed during leaf growth, whereas branching was enhanced. Consequently, highly branched areas with vigorous secondary roots alternated along the taproot with poorly branched areas with shorter roots. Root types were not affected to the same degree by shoot competition: during leaf expansion, taproot growth was just depressed but remained continuous, the emergence and elongation rates of secondary roots were significantly affected and the elongation rates of tertiary roots fell to zero. These results were consistent with the hypothesis that root growth is related to competition for assimilates and to the sink strength of the different root types, whereas root branching appeared to be promoted by leaf development.     

Secondary plasmodesmata are specific sites of localization of the tobacco mosaic virus movement protein in transgenic tobacco plants.

Expression of the tobacco mosaic virus 30-kD movement protein (TMV MP) gene in tobacco plants increases the plasmodesmatal size exclusion limit (SEL) 10-fold between mesophyll cells in mature leaves. In the present study, we examined the structure of plasmodesmata as a function of leaf development. In young leaves of 30-kD TMV MP transgenic (line 274) and vector control (line 306) plants, almost all plasmodesmata were primary in nature. In both plant lines, secondary plasmodesmata were formed, in a basipetal pattern, as the leaves underwent expansion growth. Ultrastructural and immunolabeling studies demonstrated that in line 274 the TMV MP accumulated predominantly in secondary plasmodesmata of nonvascular tissues and was associated with a filamentous material. A developmental progression was detected in terms of the presence of TMV MP; all secondary plasmodesmata in the tip of the fourth leaf contained TMV MP in association with the filamentous material. Dye-coupling experiments demonstrated that the TMV MP-induced increase in plasmodesmatal SEL could be routinely detected in the tip of the fourth leaf, but was restricted to mesophyll and bundle sheath cells. These findings are discussed with respect to the structure and function of plasmodesmata, particularly those aspects related to virus movement.     

Development patterns of telomerase activity in barley and maize

Eukaryotic chromosomes terminate with specialized structures called telomeres. Maintenance of chromosomal ends in most eukaryotes studied to date requires a specialized enzyme, telomerase. Telomerase has been shown to be developmentally regulated in man and a few other multicellular organisms, while it is constitutively expressed in unicellular eukaryotes. Recently, we demonstrated telomerase activity in plant extracts using the PCR-based TRAP (Telomeric Repeat Amplification Protocol) assay developed for human cells. Here we report telomerase activities in two grass species, barley and maize, using a modified, semi-quantitative TRAP assay. Telomerase was highly active in very young immature embryos and gradually declined during embryo development. The endosperm telomerase activity was detectable, but significantly lower than in the embryo and declined during kernel development with no detectable activity in later stages. Telomerase activity in dissected maize embryo axis was several orders of magnitude higher than in the scutellum. Telomerase activity was not detected in a range of differentiated tissues including those with active meristems such as root tips as well as the internode and leaf base. The role of telomerase repression during differentiation and the relationship between chromosome healing and telomerase activity is discussed.     

Cellular re-distribution of flavin-containing polyamine oxidase in differentiating root and mesocotyl of <Emphasis Type="Italic">Zea mays</Emphasis> L. seedlings

Plant polyamine oxidases (PAOs; EC 1.5.3.11) are hydrogen peroxide-producing enzymes supposedly involved in cell-wall differentiation processes and defence responses. Maize (Zea mays L.) PAO (MPAO) is a 53 kDa secretory glycoprotein, abundant in primary and secondary cell walls of several tissues. Using biochemical, histochemical, ultrastructural and immunocytochemical techniques, the distribution and sub-cellular compartmentalisation of MPAO in the primary root and mesocotyl of seedlings at different maturation stages or after growth under varying light conditions were analysed. In apical root tissues, MPAO immunoreactivity was mainly detected in the cytoplasmic compartment, while a lower immunoreactivity was observed in the cell walls. In the more mature, basal part of the root, intense immunogold labelling was found in the primary and secondary walls of protoxylem precursors and vessels, while endodermal cells and living metaxylem precursors were immunopositive both in their walls and in their thin cytoplasmic compartments. A re-distribution of MPAO protein from the cytoplasm toward the primary and secondary walls was also recognised when immunoreactivity of basal root tissues from 3-day-old seedlings was compared with that detected in 11-day-old tissues. Accordingly, biochemical analyses revealed MPAO entrapment in the extracellular matrix of mature tissues. In the mesocotyl, an enrichment of MPAO immunolabelling in the cell wall of protoxylem, metaxylem and epidermal tissues, as a function of light exposure, was observed. Taken together, these data support the hypothesised role of PAOs in cell-wall maturation. Moreover, the relevant intraprotoplasmic MPAO localisation observed mainly in differentiating root tissues suggests an additional role in intracellular production of hydrogen peroxide.Alessandra Cona and Sandra Moreno have contributed equally to this paper.     

4-Hydroxybenzyl alcohol accumulates in flowers and developing fruits of carrot and inhibits seed formation

Somatic embryogenesis in carrot (Daucus carota) is autonomously inhibited by 4-hydroxybenzyl alcohol (4HBA), which is produced by embryogenic cells. Because somatic embryogenesis is used as a model of zygotic embryogenesis, we assayed for 4HBA in carrot seeds and analyzed the effect of 4HBA on seed formation to determine whether 4HBA is also produced during zygotic embryogenesis. HPLC analysis showed that 4HBA accumulated in flowers and immature and mature fruits, but not in vegetative tissues. The concentration of 4HBA was highest after flowering, when the zygote developed into the early globular-stage embryo. 4HBA accumulation then decreased with seed development. Exogenous application of 4HBA to immature carrot fruits inhibited seed formation. Many 4HBA-treated seeds did not include a mature embryo. These results indicate that the production and accumulation of 4HBA occurs during carrot seed development and that 4HBA has an inhibitory effect on carrot seed formation.     

Tight regulation of expression of two Arabidopsis cytosolic Hsp90 genes during embryo development

The spatial and temporal distribution of expression of two cytosolic members of the AtHsp90 gene family was assessed during early development. In stressed transgenic plants bearing the AtHsp90-3 promoter, beta-glucuronidase (GUS) activity was strong in meristematic tissues. Expression was also detected in vascular tissues, leaf veins, siliques, and in pollen sacs. The promoter induced gene expression after heat shock in a time-course dependent manner. AtHsp90-1 promoter activity was low throughout the early stages of embryo development but high just before embryo maturation, with expression most prominent in cotyledons. AtHsp90-3 promoter activity was almost constant and restricted to the root and the cotyledon tips of the embryo. This highly specific spatial distribution of GUS activity changed when the tissues were heat-stressed. Both promoters were also active in unstressed mature pollen grains and during pollen germination. The results shown here indicate that different regulatory and developmental mechanisms control and differentiate the expression of the two cytosolic members of the Arabidopsis AtHsp90 gene family under normal conditions. The developmental and restricted pattern of expression of the AtHsp90-1 and -3 gene promoters in unstressed transgenic plants suggest prominent and distinctive roles of these two genes during different developmental processes.     

泽漆营养器官发育解剖学研究

采用石蜡切片法、半薄切片法对泽漆营养器官的发育过程进行了观察,同时对3种器官中乳汁管的分布和大小进行了分析。结果表明:泽漆根的发育类似于草本双子叶植物根的一般发育规律。初生木质部为三原型。茎的初生结构由表皮、皮层、维管束环和髓构成,其髓中有空腔,而茎的次生生长过程中维管形成层的活动短暂,仅产生少量次生维管组织,不形成周皮。叶的发育包括原分生组织、初生分生组织和成熟结构3个阶段,属于异面叶结构。泽漆乳汁管主要分布在维管束韧皮部的外侧。在3种器官中,乳汁管直径差异较大,依次为根>茎>叶。     

The Development and Structure of the Late Embryo in Torreya grandis

The materials used in this investigation were collected during 1980–1983 from Zhuji county of Zhejiang province, China. Seed of Torrcya grandis is an important dry “fruit” and used for edible oil. It is endemic to China. The primordia of male strobili are differentiated before October in the first year, while those of female strobili occur later. The microspore mother cells and megaspore mother cells are found in March and April in the second year respectively. The fertilization takes place in August and the dormant embryo overwinters at the proembryo stage. Eventually the proembryo begins to differentiate and its development starts in July of the third year. Thus the interval from fertilization to latembryogeny of Torreya grandis lasts for about 11 months. When the seeds of Torreya grandis are shed 'in August the embryo within the seed is still immature. It requires a period of after-ripening. The experiments show that the embryo resumes to develop and differentiate during 1–3 months in stratification in moist sands. The development and structure of late embryo are characterized as follows: 1. The cotyledon of the mature embryo in Torreya grandis is of 15000 μm in length and 87% of the embryo. The hypocotyl is vary shert and only 13% of the embryo. This kind of structure of the embryo in Torreya is very rare among conifers and in some degree similar to that of Keteleeria. When seed is shed the meristem of cotyledon is just differentiated and only 100–200 μm in length at the end of July to the middle of August. As the seeds are stratified in moist sands for 1–3 months, the cotyledon increases about 100 times than in room temperature in Zhuji county. 2. There is a large secretory canal in either side between the procambium and the cortex of the mature embryo. The secretory canal consists of epithelial cells of 4–5 layers. It is very peculiar in conifers. 3. The shoot apex does not begin to differentiate, until the seed has been fallen from the tree. 4. The column of the root cap is rather short and consists of the cells of about 10 layers in height and 6 layers in width. 5. Proteins are only found in the focal zone of the free apex of the young embryo but without any starch grains. The starch is abundantly distributed in the opposite end from the root initials down to root cap and the entire transitional zone. It is interesting to note that neither proteins nor starch grains are found in the suspensor system. It is assumed that the protein may be the main form of storing material in the actively growing cells and tissues of embryo in Torreya grandis.     

六盘山鸡爪大黄根蒽醌类化合物组织化学定位的研究

采用组织化学方法研究了六盘山鸡爪大黄根蒽醌类化合物的组织化学定位特征及贮藏和积累的规律。结果表明：蒽醌类化合物在根内的贮藏是多位点的,在根周皮的木栓层和栓内层、次生维管组织的维管射线和根中央的部分木薄壁细胞内不同程度地贮藏和积累了一定数量的蒽醌类化合物,次生木质部的木射线和次生韧皮部的韧皮射线是主要贮藏和积累的部位,早期形成的维管射线中蒽醌类化合物的含量较晚期形成的射线含量高。     

DEVELOPMENTAL ANATOMY IN ROOTS OF IPOMOEA PURPUREA. II. INITIATION AND DEVELOPMENT OF SECONDARY ROOTS

In seedlings of Ipomoea purpurea secondary roots are initiated in the primary root pericycle opposite immature protoxylem. Cells derived from immature endodermis, pericycle, and incipient protoxylem and stelar parenchyma contribute to the primordium. The derivatives of the endodermis become a uniseriate covering over the tip and flanks of the primordium and emerged secondary root; the endodermal covering is sloughed off when the lateral root reaches 1–5 mm in length. A series of periclinal and anticlinal divisions in the pericycle and its derivatives gives rise to the main body of the secondary root. The initials for the vascular cylinder, cortex, and rootcap-epidermis complex are established very early during primordium enlargement. After emergence from the primary root, the cortical initials undergo significant structural modifications related to enlargement of the ground meristem and cortex, and the rootcapepidermal initials are partitioned into columellar initials and lateral rootcapepidermal initials. Procambium diameter increases by periclinal divisions in peripheral sectors. The mature vascular cylinder is comprised of several vascular patterns, ranging from diarch to pentarch, that are probably related ontogenetically. Cells derived from incipient protoxylem and stelar parenchyma cells of the primary root form the vascuar connection between primary and secondary roots.