A Change in Tubulin Synthesis in the Process of Tracheary Element Differentiation and Cell Division of Isolated Zinnia Mesophyll Cells

Changes in tubulin synthesis in the process of cytodifferentiationinto tracheary elements and cell division were investigatedusing a culture of single cells isolated from the mesophyllof Zinnia elegans. The tubulin content was measured by a sensitiveimmunoblotting method using a mouse monoclonal antibody to -or ß-tubulin as a probe and mung bean tubulin as astandard. Freshly isolated mesophyll cells had only small amountsof tubulin, but the content increased rapidly between 24 and48 h of culture before morphological differentiation and celldivision. The content rose more than sixfold during 48 h cultureand then decreased slightly. This pattern of increase closelyresembled that of the increase in cortical microtubules (MTs)estimated by electron microscopic analysis. The - and ß-tubulincontents in the cultured cells were almost the same and changedin coordination during culture. The activity of tubulin synthesis was determined by densitometricscanning of spots corresponding to tubulin subunits on an autoradiogramof a two-dimensional polyacrylamide gel of [³⁵S]-methionine-labeledproteins. Tubulin synthesis began as early as between 4 and8 h of culture and its rate increased similarly to the increasein the tubulin content, with the former always preceding thelatter, indicating that the increase in content resulted fromnew tubulin synthesis. (Received December 16, 1986; Accepted February 25, 1987)     

Proteinase Activity during Tracheary Element Differentiation in Zinnia Mesophyll Cultures

The zinnia (Zinnia elegans) mesophyll cell culture tracheary element (TE) system was used to study proteinases active during developmentally programmed cell death. Substrate-impregnated gels and single-cell assays revealed high levels of proteinase activity in differentiating TEs compared with undifferentiated cultured cells and expanding leaves. Three proteinases (145, 28, and 24 kD) were exclusive to differentiating TEs. A fourth proteinase (59 kD), although detected in extracts from all tissues examined, was most active in differentiating TEs. The 28- and 24-kD proteinases were inhibited by thiol proteinase inhibitors, leupeptin, and N-[N-(L-3-trans-carboxirane-2-carbonyl)-L-leucyl]-agmatine (E-64). The 145- and 59-kD proteinases were inhibited by the serine proteinase inhibitor phenylmethylsulfonyl fluoride (PMSF). Extracts from the TE cultures contained sodium dodecyl sulfate-stimulated proteolytic activity not detected in control cultures. Sodium dodecyl sulfate-stimulated proteolysis was inhibited by leupeptin or E-64, but not by PMSF. Other tissues, sucrose-starved cells and cotyledons, that contain high levels of proteolytic activity did not contain TE-specific proteinases, but did contain higher levels of E-64-sensitive activities migrating as 36- to 31-kD enzymes and as a PMSF-sensitive 66-kD proteinase.     

Cell Expansion and Tracheary Element Differentiation Are Regulated by Extracellular pH in Mesophyll Cultures of Zinnia elegans L

The effects of medium pH on cell expansion and tracheary element (TE) differentiation were investigated in differentiating mesophyll suspension cultures of Zinnia elegans L. In unbuffered cultures initially adjusted to pH 5.5, the medium pH fluctuated reproducibly, decreasing about 1 unit prior to the onset of TE differentiation and then increasing when the initiation of new Tes was complete. Elimination of large pH fluctuations by buffering the culture medium with 20 mM 2-(N-morpholino)ethanesulfonic acid altered both cell expansion and TE differentiation, whereas altering the starting pH of unbuffered culture medium had no effect on either process. Cell expansion in buffered cultures was pH dependent with an optimum of 5.5 to 6.0. The direction of cell expansion was also pH dependent in buffered cultures. Cells elongated at pH 5.5 to 6.0, whereas isodiametric cell expansion was predominant at pH 6.5 to 7.0. The onset of TE differentiation was delayed when the pH was buffered higher or lower than 5.0. However, TEs eventually appeared in cultures buffered at pH 6.5 to 7.0, indicating that a decrease in pH to 5.0 is not necessary for differentiation. Very large TEs with secondary cell wall thickenings resembling metaxylem differentiated in cultures buffered at pH 5.5 to 6.0, which also showed the greatest cell expansion. The correlation between cell expansion and delayed differentiation of large, metaxylem-like TEs may indicate a link between the regulatory mechanisms controlling cell expansion and TE differentiation.     

Changes in the Synthesis of RNA and Protein during Tracheary Element Differentiation in Single Cells Isolated from the Mesophyll of Zinnia elegans

Cycloheximide (CH) prevented tracheary element (TE) differentiationand cell division in a culture of single cells isolated fromthe mesophyll of Zinnia elegans at the concentrations whichinhibited incorporation of [¹⁴C]-leucine into protein. Whenthe cells were pulse-treated with this inhibitor for 12 h atvarious times of culture, TE formation was inhibited most stronglyby the treatments made between 24 and 60 h of culture. Incorporationof [¹⁴C]-leucine into protein showed a high level during thisperiod. The inhibitory effect of actinomycin D (Act-D) on TEdifferentiation was also marked when it was administered from24 to 60 h of culture when incorporation of [¹⁴C]-uridine intonucleic acid was at a high level. These results indicate thatRNA and protein syntheses are prerequisites for cytodifferentiationto TE and that the syntheses between 24 and 60 h of cultureare closely associated with cytodifferentiation. Studies of qualitative changes in proteins using two-dimensionalelectrophoresis revealed that approximately 400 polypeptidesextracted from [³⁵S]-methionine-labeled cells could be reproduciblyresolved and that most of them were synthesized in both differentiatingand non-differentiating cells. During TE differentiation, however,the synthesis of two polypeptides was shut off and two otherpolypeptides were newly synthesized between 48 and 60 h of culture,preceding the morphological changes. The relationship betweenTE differentiation and the synthesis of RNA and protein is discussed. (Received November 20, 1982; Accepted February 18, 1983)     

Xylan Synthase Activity in Isolated Mesophyll Cells of Zinnia elegans during Differentiation to Tracheary Elements

A paniculate fraction obtained from mesophyll cells of Zinniaelegans that were differentiating into tracheary elements exhibitedxylan synthase activity, catalyzing the transfer of ^MC-xylosefrom UDP-D-[U-¹⁴C]-xylose into 1,4-linked xylan. The activityincreased transiently at the same time as thickening of secondarycell walls occurred, a process that is accompanied by the depositionof cellulose, xylan and lignin. (Received August 3, 1990; Accepted December 6, 1990)     

Establishment of an Experimental System for the Study of Tracheary Element Differentiation from Single Cells Isolated from the Mesophyll of Zinnia elegans

Single cells were isolated mechanically from the mesophyll of adult plants and of seedlings of Zinnia elegans L. cv. Canary bird. When single cells isolated from the first leaves of seedlings were cultured in a liquid medium in the dark with rotation, they differentiated to tracheary elements with a reasonable degree of synchrony in the 24-hour period between days 2 and 3 after culture. The proportion of tracheary elements as a percentage of total cells reached nearly 30% 3 days after culture. Factors favoring cytodifferentiation were certain optimum levels of both α-naphthalene-acetic acid (0.1 milligram per liter) and benzyladenine (1 milligram per liter), a low concentration of ammonium chloride (0 to 1 millimolar), and an initial cell population density in the range 0.4 to 3.8 × 10⁵ cells/ml. It was possible to follow analytically the sequence of cytodifferentiation in individual cells in this system.     

百日草游离叶肉细胞导管分子分化过程中过氧化物酶的催化特性

百日草游离对肉细胞随着导管分子的分化,木质素含量逐渐增加;ＰＯⅠ（可溶性ＰＯ）、ＰＯⅡ（与细胞壁离子型结合的ＰＯ）和ＰＯⅢ（与细胞壁共价结合的ＰＯ）活性增加,并分别对底物愈创木酚、丁子香酚和咖啡酸（含阿魏酸）有较大的亲和力;抑制剂对ＰＯ活性抑制的动力学特性表明．间苯三酚为竞争性抑制剂,硫酸亚铁铵和二硫苏糖醇是非竞争性抑制剂。     

Kinetics of Determination in the Differentiation of Isolated Mesophyll Cells of Zinnia elegans to Tracheary Elements

Mechanically isolated mesophyll cells of Zinnia elegans L. cv Envy differentiate to tracheary elements when cultured in inductive medium containing 0.5 micromolar α-naphthaleneacetic acid and 0.5 micromolar benzyladenine. The cells do not differentiate when cultured in medium in which the concentration of auxin and/or cytokinin has been reduced to 0.005 micromolar. Cells require an initial 24-hour exposure to inductive cytokinin and 56-hour exposure to inductive auxin for differentiation at 72 hours of culture. Freshly isolated Zinnia cells can be maintained in medium having low concentrations of both auxin and cytokinin for only 1 day without significant loss of potential to differentiate upon transfer to inductive medium. Initial culture for up to 2 days in medium having high auxin and low cytokinin, or low auxin and high cytokinin, allows full differentiation on the third day after transfer to inductive medium and potentiates the early differentiation of some cells.     

Effect of Inhibitors of ADP-ribosyltransferase on the Differentiation of Tracheary Elements from Isolated Mesophyll Cells of Zinnia elegans

The effect of m-aminobenzamide (m-ABm) and nicotinamide, inhibitorsof ADP-ribosyltransferase (ADP-RT), on the differentiation oftracheary elements was investigated using isolated mesophyllcells of Zinnia elegans. Both compounds inhibited differentiationwithout affecting cell division, a result which suggests theinvolvement of ADP-RT. The effects of thymidine, a potent inhibitorof ADP-RT and isomers of m-ABm were also examined. (Received September 1, 1986; Accepted January 29, 1987)     

Imaging Cell Wall Architecture in Single Zinnia elegans Tracheary Elements

The chemical and structural organization of the plant cell wall was examined in Zinnia elegans tracheary elements (TEs), which specialize by developing prominent secondary wall thickenings underlying the primary wall during xylogenesis in vitro. Three imaging platforms were used in conjunction with chemical extraction of wall components to investigate the composition and structure of single Zinnia TEs. Using fluorescence microscopy with a green fluorescent protein-tagged Clostridium thermocellum family 3 carbohydrate-binding module specific for crystalline cellulose, we found that cellulose accessibility and binding in TEs increased significantly following an acidified chlorite treatment. Examination of chemical composition by synchrotron radiation-based Fourier-transform infrared spectromicroscopy indicated a loss of lignin and a modest loss of other polysaccharides in treated TEs. Atomic force microscopy was used to extensively characterize the topography of cell wall surfaces in TEs, revealing an outer granular matrix covering the underlying meshwork of cellulose fibrils. The internal organization of TEs was determined using secondary wall fragments generated by sonication. Atomic force microscopy revealed that the resulting rings, spirals, and reticulate structures were composed of fibrils arranged in parallel. Based on these combined results, we generated an architectural model of Zinnia TEs composed of three layers: an outermost granular layer, a middle primary wall composed of a meshwork of cellulose fibrils, and inner secondary wall thickenings containing parallel cellulose fibrils. In addition to insights in plant biology, studies using Zinnia TEs could prove especially productive in assessing cell wall responses to enzymatic and microbial degradation, thus aiding current efforts in lignocellulosic biofuel production.The organization and molecular architecture of plant cell walls represent some of the most challenging problems in plant biology. Although much is known about general aspects of assembly and biosynthesis of the plant cell wall, the detailed three-dimensional molecular cell wall structure remains poorly understood. The highly complex and dynamic nature of the plant cell wall has perhaps limited the generation of such detailed structural models. This information is pivotal for the successful implementation of novel approaches for conversion of biomass to liquid biofuels, given that one of the critical processing steps in biomass conversion involves systematic deconstruction of cell walls. Therefore, a comprehensive understanding of the architecture and chemical composition of the plant cell wall will not only help develop molecular-scale models, but will also help improve the efficiency of biomass deconstruction.The composition and molecular organization of the cell wall is species and cell type dependent (Vorwerk et al., 2004). Thus, the development of a model plant system, which utilizes a single cell type, has enhanced our capacity to understand cell wall architecture. The ability to generate a population of single Zinnia elegans plant cells that were synchronized throughout cell wall deposition during xylogenesis was developed in the 1980s (Fukuda and Komamine, 1980). Mesophyll cells isolated from the leaves of Zinnia and cultured in the presence of phytohormones will transdifferentiate into tracheary elements (TEs), which are individual components of the xylem vascular tissue (Fukuda and Komamine, 1980). During this transdifferentiation process, TEs gradually develop patterned secondary wall thickenings, commonly achieving annular, spiral, reticulate, scalariform, and pitted patterns (Bierhorst, 1960; Falconer and Seagull, 1988; Roberts and Haigler, 1994). These secondary wall thickenings serve as structural reinforcements that add strength and rigidity to prevent the collapse of the xylem under the high pressure created by fluid transport. During the final stages of transdifferentiation, TEs accumulate lignin in their secondary walls and undergo programmed cell death, which results in the removal of all cell contents, leaving behind a “functional corpse” (Roberts and McCann, 2000; Fukuda, 2004).In broad terms, the primary cell wall of higher plants is mainly composed of three types of polysaccharides: cellulose, hemicelluloses, and pectins (Cosgrove, 2005). Cellulose is composed of unbranched β-1,4-Glc chains that are packed together into fibrils by intermolecular and intramolecular hydrogen bonding. Hemicelluloses and pectins are groups of complex polysaccharides that are primarily composed of xyloglucans/xylans and galacturonans, respectively. Hemicelluloses are involved in cross-linking and associating with cellulose microfibrils, while pectins control wall porosity and help bind neighboring cells together. The patterned deposits of secondary wall in Zinnia TEs primarily consist of cellulose microfibrils, along with hemicelluloses, and also lignin, a complex aromatic polymer that is characteristic of secondary walls and provides reinforcement (Turner et al., 2007). All the molecular components in the cell wall correspond to a multitude of different polysaccharides, phenolic compounds, and proteins that become arranged and modified in muro, yielding a structure of great strength and resistance to degradation.Currently, electron microscopy is the primary tool for structural studies of cell walls and has provided remarkable information regarding wall organization. Fast-freeze deep-etch electron microscopy in combination with chemical and enzymatic approaches have generated recent models of the architecture of the primary wall (McCann et al., 1990; Carpita and Gibeaut, 1993; Nakashima et al., 1997; Fujino et al., 2000; Somerville et al., 2004). Direct visualization of secondary wall organization has been focused toward the examination of multiple wall layers in wood cells (Fahlen and Salmen, 2005; Zimmermann et al., 2006). However, few studies have examined the secondary wall, so our knowledge regarding the higher order architecture of this type of wall is limited. Over the past few decades, atomic force microscopy (AFM) has provided new opportunities to probe biological systems with spatial resolution similar to electron microscopy techniques (Kuznetsov et al., 1997; Muller et al., 1999), with additional ease of sample preparation and the capability to probe living native structures. AFM has been successfully applied to studies of the high-resolution architecture, assembly, and structural dynamics of a wide range of biological systems (Hoh et al., 1991; Crawford et al., 2001; Malkin et al., 2003; Plomp et al., 2007), thus enabling the observation of the ultrastructure of the plant cell wall, which is of particular interest to us (Kirby et al., 1996; Morris et al., 1997; Davies and Harris, 2003; Yan et al., 2004; Ding and Himmel, 2006).To generate more detailed structural models, knowledge about the structural organization of the cell wall can be combined with spatial information about chemical composition. Instead of utilizing chromatography techniques to analyze cell wall composition by extracting material from bulk plant samples (Mellerowicz et al., 2001; Pauly and Keegstra, 2008), Fourier transform infrared (FTIR) spectromicroscopy can be used to directly probe for polysaccharide and aromatic molecules in native as well as treated plant material (Carpita et al., 2001; McCann et al., 2001). FTIR spectromicroscopy is not only able to identify chemical components in a specific system but also can determine their distribution and relative abundance. This technique also improves the sensitivity and spatial resolution of cellular components without the derivatization needed by chemical analysis using chromatography. Polysaccharide-specific probes, such as carbohydrate-binding modules (CBMs), can also be used to understand the chemical composition of the plant cell wall. CBMs are noncatalytic protein domains existing in many glycoside hydrolases. Based on their binding specificities, CBMs are generally categorized into three groups: surface-binding CBMs specific for insoluble cellulose surfaces, chain-binding CBMs specific for single chains of polysaccharides, and end-binding CBMs specific for the ends of polysaccharides or oligosaccharides. A surface-binding CBM with high affinity for the planar faces of crystalline cellulose (Tormo et al., 1996; Lehtio et al., 2003) has been fluorescently labeled and used to label crystals as well as plant tissue (Ding et al., 2006; Porter et al., 2007; Liu et al., 2009; Xu et al., 2009). The binding capacity of the CBM family has been further exploited for the detection of different polysaccharides, such as xylans and glucans, and can thus be used for the characterization of plant cell wall composition (McCartney et al., 2004, 2006).In this study, we used a combination of AFM, synchrotron radiation-based (SR)-FTIR spectromicroscopy, and fluorescence microscopy using a cellulose-specific CBM to probe the cell wall of Zinnia TEs. The Zinnia TE culture system proved ideal for observing the structure and chemical composition of the cell wall because it comprises a single homogeneous cell type, representing a simpler system compared with plant tissues, which may contain multiple cell types. Zinnia TEs were also advantageous because they were analyzed individually, and population statistics were generated based on specific conditions. Furthermore, cultured Zinnia TEs were used for the consistent production of cell wall fragments for analysis of the organization of internal secondary wall structures. In summary, we have physically and chemically dissected Zinnia TEs using a combination of imaging techniques that revealed primary and secondary wall structures and enabled the reconstruction of TE cell wall architecture.     

Secondary Cell Wall Formation: Changes in Cell Wall Constituents during the Differentiation of Isolated Mesophyll Cells of Zinnia elegans to Tracheary Elements

Changes in the cell walls and their sugar composition duringthe formation of tracheary elements (TE) were analyzed usinga culture of single cells isolated from the mesophyll of Zinniaelegans. By using Calcofluor White the first differentiatingcells were observed 36 to 38 h after the start of culture. Thisis 8 to 10 hours before differentiating cells can be observedwithout staining, and about 14 to 16 hours before the beginningof lignification of differentiating cells. In correlation withthe appearance of differentiating cells, the following changeswere observed: (1) a significant increase in the total carbohydratein the 5% KOH-soluble, the 24% KOH-soluble and insoluble cellulosicfractions; (2) a decrease in the relative amount of uronic acidsin the EDTA-soluble fraction which corresponded to increasesin the KOH-soluble fractions and in the insoluble fraction;(3) an enormous increase in the absolute and relative amountof xylose in the hemicellulosic fractions and to some extentalso in the cellulosic fraction. Methylation analysis indicatedthat the high amount of xylose reflects the synthesis of a xylan-typepolysaccharide which is deposited simultaneously with celluloseprior to the lignification of the wall. (Received August 5, 1987; Accepted December 9, 1987)     

Relationship between Tracheary Element Differentiation and DNA Synthesis in Single Cells Isolated from the Mesophyll of Zinnia elegans--Analysis by Inhibitors of DNA Synthesis

Effects of inhibitors of DNA synthesis on tracheary element(TE) differentiation were investigated in a culture of singlecells isolated from the mesophyll of Zinnia elegans L. cv. Canarybird. In this system, neither mitosis nor replication of thewhole genome during the S phase in the cell cycle is a prerequisitefor TE differentiation [Fukuda and Komamine (1980) Plant Physiol.65: 61, unpublished data]. Fluorouracil (FU), fluorodeoxyuridine(FUdR), mitomycin G (MC), arabinosyl cytosine (ara-C) and aphidicolin,inhibitors of DNA synthesis, prevented the incorporation of[³H]-thymidine into nucleic acid, cell division and cytodifferentiationto TE. However, neither FUdR nor aphidicolin prevented the incorporationof [14C]-leucine into protein. Thymidine reversed the inhibitoryeffect of FUdR when given simultaneously with FUdR. These resultsshow that the inhibitors of DNA synthesis prevent TE differentiationvia blockage of the synthesis of some DNA, although replicationof the whole genome during the S phase is not a prerequisitefor cytodifferentiation. The role of DNA synthesis in TE differentiationis discussed. (Received October 13, 1980; Accepted November 17, 1980)     

Proteasome Inhibitors Prevent Tracheary Element Differentiation in Zinnia Mesophyll Cell Cultures

To determine whether proteasome activity is required for tracheary element (TE) differentiation, the proteasome inhibitors clasto-lactacystin β-lactone and carbobenzoxy-leucinyl-leucinyl-leucinal (LLL) were used in a zinnia (Zinnia elegans) mesophyll cell culture system. The addition of proteasome inhibitors at the time of culture initiation prevented differentiation otherwise detectable at 96 h. Inhibition of the proteasome at 48 h, after cellular commitment to differentiation, did not alter the final percentage of TEs compared with controls. However, proteasome inhibition at 48 h delayed the differentiation process by approximately 24 h, as indicated by examination of both morphological markers and the expression of putative autolytic proteases. These results indicate that proteasome function is required both for induction of TE differentiation and for progression of the TE program in committed cells. Treatment at 48 h with LLL but not clasto-lactacystin β-lactone resulted in partial uncoupling of autolysis from differentiation. Results from gel analysis of protease activity suggested that the observed incomplete autolysis was due to the ability of LLL to inhibit TE cysteine proteases.     

A Protease Activity Displaying Some Thrombin-like Characteristics in Conditioned Medium of Zinnia Mesophyll Cells Undergoing Tracheary Element Differentiation

The normal development of tracheary elements (TE) requires a selective degradation of the cytoplasm without loss of the extracellular wall that remains behind as the water-conducting units of xylem. Using zinnia-(Zinnia elegans L. cv. Green Envy) cultured mesophyll cells that synchronously transdifferentiate into TEs, extracellular and intracellular proteases, respectively, have been shown to both trigger death and to execute autolysis as the final component of a programmed cell death (PCD). We report here the appearance in the medium of an unusual proteolytic activity correlated with the PCD process just prior to the autolysis. The activity has a pH optimum of 5.5–6.0 and displays some thrombin characteristics. This protease activity has 1) a 10-fold higher affinity towards a thrombin-specific chromogenic substrate than toward a trypsin-specific chromogenic substrate; 2) a 1000-fold lower sensitivity to soybean trypsin inhibitor (STI) compared to trypsin; and 3) limited ability to cleave the protease-activated receptor-1, the native thrombin substrate. However, the addition of partially purified fraction containing the thrombin-like protease activity to the medium of PCD-competent cells does not prematurely trigger PCD, and the thrombin-specific peptide inhibitor phenylalanine-proline-aspartic acid-chloromethylketone fails to inhibit PCD or tracheary element (TE) formation. This suggests that this protease activity may play a role within the cells in execution of the autolysis or in the collapse of the tonoplast rather than as an extracellular proteolytic activity participating in the chain of events leading to cell death. Online publication: 7 April 2005     

Direct Evidence for Cytodifferentiation to Tracheary Elements without Intervening Mitosis in a Culture of Single Cells Isolated from the Mesophyll of Zinnia elegans

A serial observation of the process of tracheary element differentiation from single cells isolated from the mesophyll of Zinnia elegans L. cv. Canary bird provided the first direct evidence for the cytodifferentiation without intervening mitosis. Percentage of the tracheary elements formed without cell division was about 60% of total tracheary elements formed on the 4th day of culture. The number of tracheary elements formed without intervening mitosis was not reduced in the presence of colchicine at the concentrations blocking cell division. These facts clearly indicate that cell division is not a prerequisite for tracheary element differentiation in this system.     

Suppression by 5-Bromo-2'-deoxyuridine of Transdifferentiation into Tracheary Elements of Isolated Mesophyll Cells of Zinnia elegans

5-Bromo-2'-deoxyuridine (BrdU), a thymidine analog, suppressedthe transdifferentiation into tracheary elements (TE) of isolatedmesophyll cells of Zinnia elegans without affecting cell division.Tracer experiments with [³H]BrdU indicated that 76% and 24%of the incorporated radioactivity was located in the DNA andthe RNA, respectively. Both thymidine and uridine counteractedthe inhibitory effect of BrdU on transdifferentiation but thymidinewas much more effective than uridine. These results suggestthat BrdU might interfere with transdifferentiation via itsincorporation into DNA. The timing of effective suppressionby BrdU was examined by monitoring the effects of the sequentialaddition of BrdU and thymidine with an interval of 12 h at varioustimes in culture. Transdifferentiation was most sensitive toBrdU between the 24th and the 36th hour of culture. This resultsuggests that this window of time is critical for DNA synthesisduring the transdifferentiation of isolated mesophyll cellsof Zinnia elegans into TE. ³Present address: Department of Chemical and Biological Sciences,Faculty of Science, Japan Women's University, Mejiro, Tokyo,112 Japan     

Characteristics of the Inhibitory Effect of 5-Fluorodeoxyuridine on Cytodifferentiation into Tracheary Elements of Isolated Mesophyll Cells of Zinnia elegans

The inhibitory effect of 5-fluorodeoxyuridine (FdU) on the differentiationinto tracheary elements was characterized in isolated mesophyllcells of Zinnia elegans. Both thymidine and uridine counteracted the inhibitory effectof FdU on the differentiation into tracheary elements, whileonly thymidine was effective in counteracting the effect ofFdU on cell division. Higher concentrations of thymidine wereneeded for the restoration of the differentiation that was blockedby FdU than for the restoration of cell division. These resultssuggest that FdU prevents the differentiation via a mechanismthat is different from the inhibition of thymidylate (dTMP)synthase by fluorodeoxyuridine monophosphate (FdUMP), derivedfrom FdU, to which the blockage of cell division by FdU shouldbe attributable. The differentiation into tracheary elements was prevented whenFdU was added earlier than the 36th hour of culture, and thymidineovercame the inhibitory effect of FdU only when added withinthe first 4 h of culture. Pretreatment with FdU before applicationof 6-benzyladenine (BA) and 1-naphthaleneacetic acid (NAA),which are essential for the formation of tracheary elements,also inhibited the differentiation. Thus, the aspect of thedifferentiation that is the target of inhibition by FdU appearsto occur between the 4th hour and the 36th hour of culture andto begin even in the absence of exogenous plant growth regulators. (Received April 3, 1989; Accepted October 27, 1989)     

Characterization of the Inhibitory Effect of Aphidicolin on Transdifferentiation into Tracheary Elements of Isolated Mesophyll Cells of Zinnia elegans

Inhibition by aphidicolin (APC), an inhibitor specific for -typeDNA polymerase, of trans-differentiation into tracheary elementswas characterized in Zinnia mesophyll cells. APC was effectivewhen given in the first 24 h of culture and exposure continueduntil the 36th hour. This suggests temporal involvement of -typeDNA polymerase in transdifferentiation. ¹Present address: Department of Chemical and Biological Sciences,Faculty of Science, Japan Women's University, Mejiro, Tokyo,112 Japan     

Involvement of Poly(ADP-Ribose) Synthesis in Transdifferentiation of Isolated Mesophyll Cells of Zinnia elegans into Tracheary Elements

The relationship between poly(ADP-ribose) synthesis and cytodifferentiationwas studied in the well characterized Zinnia system, in whichisolated mesophyll cells of Zinnia elegans transdifferentiateinto tracheary elements (TE) in a suspension culture in thepresence of both auxin and cytokinin. The rate of poly(ADP-ribose)synthesis was measured in nuclei isolated from cells that hadbeen induced to undergo transdifferentiation, and activationof such synthesis was observed before the appearance of TE duringculture. In cultures without auxin or cytokinin, poly-(ADP-ribose)synthesis appeared to proceed much more slowly. Treatment of cells with a potent inhibitor of poly-(ADP-ribose)polymerase, namely, 6(5H)-phenanthridinone (PT), resulted inthe blockage of TE formation and a decrease in the frequencyof cell division. PT was very effective in interfering withtransdifferentiation, in particular, when supplied between the24th hour and the 36th hour of culture. Repair-type DNA synthesis,which has been proposed to participate in transdifferentiation,was suppressed by the treatment with PT. These results suggestthat poIy(ADP-ribose) synthesis and subsequent repair-type DNAsynthesis might play a critical role in the transdifferentiationof Zinnia cells. ³Present address: Botanical Gardens, Faculty of Science, Universityof Tokyo, Hakusan, Bunkyo-ku, Tokyo, 112 Japan. ⁴Present address: Department of Chemical and Biological Sciences,Faculty of Science, Japan Women's University, Mejirodai, Bunkyo-ku,Tokyo, 112 Japan.     

Inhibition of Cell Division and DNA Synthesis by Gibberellin in Isolated Zinnia Mesophyll Cells

A culture system of isolated mesophyll cells of Zinnia eleganswas used to examine the action of gibberellic acid (GA) on celldivision. Isolated Zinnia mesophyll cells cultured in a mediumcontaining auxin and cytokinin reinitiated cell division ina partly synchronized manner. When mesophyll cells isolatedfrom 21-day-old seedlings were used, GA added to the culturemedium at concentrations of 1 x 10^–6 M or higher suppressedthe initial rise in the number of divided cells. Tracer experimentswith [³H]-dThd revealed that GA treatment inhibited the incorporationof [³H]-dThd into DNA in the nucleus without inhibiting theuptake of [³H]-dThd into the cells, indicating that GA inhibitedDNA synthesis. GA applied at 48 h inhibited the incorporationof [³H]-dThd into DNA during the following 24 h, but GA appliedat 72 h did not inhibit the incorporation during the subsequent24 h. This suggests that GA affects the process of reinitiationof DNA synthesis, but does not affect DNA synthesis once cellshave become proliferative. (Received January 14, 1986; Accepted March 31, 1986)