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)     

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。     

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)     

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)     

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.     

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)     

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.     

Effects of Nutrient Limitation and {gamma}-Irradiation on Tracheary Element Differentiation and Cell Division in Single Mesophyll Cells of Zinnia elegans

The effects of nutrient limitation and -irradiation on trachearyelement differentiation and cell division were investigatedusing single cells isolated from the mesophyll of Zinnia elegans.When the phosphate concentration of the medium was reduced to10 µM (1/50 of Fukuda and Komamine's medium, 1980a), thefrequency of cell division during 4 days of culture decreased,while the frequency of tracheary element differentiation wasunaffected. -Irradiation with a dose of 92 Gy at 36 h of culturepreferentially and thoroughly suppressed cell division withoutreducing the number of tracheary elements formed. The appearanceof secondary cell wall thickenings was delayed by irradiation,but synchrony was maintained. Thus the Zinnia system previouslyreported [Fukuda and Komamine (1980a) Plant Physiol. 65: 57]was improved to give a more useful system for the study of cytodifferentiation,in which tracheary element formation occurred from single cellswithout cell division. ¹Present address: Biological Institute, Faculty of Science,Tohoku University, Sendai, Miyagi 980, Japan. (Received November 28, 1985; Accepted February 22, 1986)     

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.     

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)     

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.     

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     

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)     

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     

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)     

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.     

Tracheary Element Formation from Single Isolated Cells in Culture

Friable callus tissue of Centaurea cyanus L. was grown on a solidified synthetic nutrient medium (EBM-1) to produce a tissue with a low frequency of differentiated tracheary elements. Tissues were then suspended in liquid nutrient medium with agitation to produce a suspension which was filtered and the single-cell suspension resulting was used as inoculum for either cell suspension cultures or for plating of cells into solidified medium in Petri plates. Media for the suspension cultures were selected to favor cytodifferentiation of tracheary elements. Differentiated tracheary elements formed as early as 10 days and numbers of tracheary elements increased with time roughly in relation to the increase in total cell number. From plating experiments it was shown conclusively that single isolated parenchyma cells differentiated directly into single isolated tracheary elements, although this event was rare. More usual was the division of isolated cells to form small colonies and then the differentiation of one, several or all of the cells into tracheary elements. Comparisons are made between results with cell plating experiments and cell suspension cultures. Optimism is expressed for finding a cell suspension culture system for studying cytodifferentiation.     

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     

Brassinosteroids Act as Regulators of Tracheary-Element Differentiation in Isolated Zinnia Mesophyll Cells

Uniconazole [S-3307; (E)-l-(4-chlorophenyl)-4,4-dimethyl-2-(l,2,4-triazol-l-yl)-l-penten-3-ol],a synthetic plant-growth retardant, inhibited the differentiationof isolated mesophyll cells of Zinnia elegans L. into trachearyelements (TEs) but had no effect on cell division when it wasadded to the culture medium at a concentration of 3.4 µM.In the presence of uniconazole, none of the cytological eventscharacteristic of the processes of TE differentiation, suchas aggregation of actin filaments, bundling of microtubulesor localized thickening and lignification of secondary walls,was observed. Uniconazole was effective when it was added tothe medium within 36 h after the start of culture. Brassinosteroids(0.2 nM brassinolide or 2 µM homobrassinolide), but notgibberellin A₃, counteracted the inhibitory effect of uniconazoleon TE differentiation. Brassinosteroids were most effectivewhen they were added to cultures between 24 and 30 h after thestart of culture. Exogenously applied brassinosteroids promotedTE differentiation. It is suggested that the synthesis of brassinosteroidsis essential for the differentiation of the cells into TEs andthat uniconazole inhibits this differentiation through its inhibitoryeffect on the biosynthesis of brassinosteroids. (Received May 9, 1991; )