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)     

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)     

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.     

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)     

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。     

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.     

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)     

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.     

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.     

Transient and Specific Expression of a Cysteine Endopeptidase Associated with Autolysis during Differentiation of Zinnia Mesophyll Cells into Tracheary Elements

Endopeptidase activities during the differentiation of Zinniacells into tracheary elements (TEs) were examined with severalpeptidyl 4-methyl-7-coumarylamido (MCA) as substrates. The activitythat hydrolysed carbobenzoxy-Phe-Arg-MCA (Z-Phe-Arg-MCA) atpH 5 increased in a differentiation-related manner: this activity,which was not observed in freshly isolated mesophyll cells wasinduced by the combination of -naphthaleneacetic acid (NAA)and 6-benzyladenine (BA) that is necessary for differentiationof TEs, but not by NAA or BA alone. The activity in cells culturedin TE-inductive medium that contained both NAA and BA increasedvery rapidly between the 48th and 60th hours of culture, whenthe number of TE increased rapidly. A protease responsible forthis activity with a molecular mass of 30 kDa was partiallypurified from cells which had been cultured in the TE-inductivemedium and included many immature TEs. Strong inhibition by[L-3-trans-carboxyoxiran-2-carbonyl]-L-Leu-agmatin (E-64), andactivation by dithiothreitol (DTT) indicated that this proteasebelongs to a family of cysteine endopeptidases (EC 3.4.22). ¹Present address: Department of Material and Biological Engineering,Tsuruoka National College of Technology, Inooka-Sawada 104,Tsuruoka, Yamagata, 997 Japan     

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; )     

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)     

Immunocytochemical Localization of Phenylalanine Ammonia-Lyase and Cinnamyl Alcohol Dehydrogenase in Differentiating Tracheary Elements Derived from Zinnia Mesophyll Cells

Secondary wall thickening is the most characteristic morphologicalfeature of the differentiation of tracheary elements. Isolatedmesophyll cells of Zinnia elegans L. cv. Canary Bird in differentiationmedium are converted to tracheary elements, which develop lignifiedsecondary wall thickenings. Using this system, we investigatedthe distribution of two enzymes, phenylalanine ammonia-Iyase(PAL) (EC 4.3.1.5 [EC] ) and cinnamyl alcohol dehydrogenase (CAD)(EC 1.1.1.195 [EC] ), by both biochemical and immunological methods.Both PAL and CAD appear to be key enzymes in the biosynthesisof lignin precursors, and they have been shown to be associatedwith the differentiation of tracheary elements. Cultured cellswere collected after various times in culture. The culture mediumwas separated from cells by centrifugation and designated fraction(1), the extracellular fraction. The collected cells were homogenizedand separated into four fractions: (2) cytosol; (3) microsomes;(4) cell walls (loosely bound material); and (5) cell walls(tightly bound material). PAL activity was detected in eachfraction. The extracellular fraction consistently had the greatestPAL activity. Moreover, PAL activity in the cytosolic fractionincreased rapidly prior to lignification, as it did in boththe microsomal and the cell wall (tightly bound) fractions duringlignification. Antisera against PAL and against CAD detectedthe proteins with molecular masses that corresponded to thoseof PAL and CAD in Zinnia. Immuno-electron microscopy revealedthat, in differentiating tracheary elements, PAL was dispersedin the cytoplasmic matrix and was located on Golgi-derived vesiclesand on the secondary wall thickenings. "Cell-free" immuno-lightmicroscopy supported the putative distribution of PAL on lignifyingsecondary walls. The pattern of distribution of CAD was similarto that of PAL. Thus, both PAL and CAD seemed to be localizedin secondary wall thickenings. From the results of both biochemicalassays and immunocytochemical staining, it appeared that atleast two types of PAL and CAD are present in differentiatingcells. One type of each enzyme is distributed in the cytosol,while the other is secreted from the Golgi apparatus and transportedby Golgi-derived vesicles to the secondary wall thickenings. (Received April 19, 1996; Accepted November 18, 1996)     

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     

4-Coumarate:Coenzyme A Ligase and Isoperoxidase Expression in Zinnia Mesophyll Cells Induced to Differentiate into Tracheary Elements

When cultured in inductive medium containing adequate auxin and cytokinin, isolated mesophyll cells of Zinnia elegans L. cv Envy differentiate into tracheary elements with lignified secondary wall thickenings. Differentiation does not occur when cells are cultured in control medium, which has reduced levels of auxin and/or cytokinin. The activities of two enzymes involved in lignin synthesis, 4-coumarate:coenzyme A ligase and peroxidase, were examined. An induction-specific cationic isoperoxidase, visualized by low pH polyacrylamide gel electrophoresis, is detectable in soluble and wall fractions of cultured Zinnia cells long before tracheary elements visibly differentiate and is thus an early marker of differentiation. Compounds (such as antiauxins, anticytokinins, and tunicamycin) that inhibit or delay differentiation alter the expression of this isoperoxidase. 4-Coumarate:coenzyme A ligase activity increases dramatically only as cells differentiate. Together, these results suggest that the onset of lignification in differentiating Zinnia cells might be controlled by the availability of precursors synthesized by way of 4-coumarate:coenzyme A ligase. These precursors would then be polymerized into lignin in the cell wall by the induction-specific isoperoxidase.     

Regulation of Tubulin Degradation in Isolated Zinnia Mesophyll Cells in Culture

Tubulin degradation in isolated Zinnia mesophyll cells in culturewas investigated by pulse-chase labeling with [³⁵S]-methionineand two-dimensional electrophoresis. Tubulin degradation changesdynamically during culture. Almost no tubulin degradation occursin the cells on the first day in culture. Treatment of thesecells with colchicine activates the degradation of tubulin,but not of proteins other than tubulin. In the presence of colchicine,the and ß-subunits of tubulin are degraded togetherand the half life of each subunit is approximately 6 h. After2 d in culture, there is active degradation of tubulin evenin the absence of colchicine. Colchicine did not inhibit new synthesis of tubulin in Zinniacells. This is very different from the results reported in culturedmammalian cells, whereby unpolymerized tubulin elevated by colchicine-treatmentdepresses its own synthesis. These and previous results dealing with changes in the leveland synthesis of tubulin in cultured Zinnia cells (Fukuda 1987),are discussed in relation to the regulation of tubulin metabolismin cultured Zinnia cells. ¹Present address: Biological Institute, Faculty of Science,Tohoku University, Aoba-yama, Sendai, 980 Japan. (Received September 5, 1988; Accepted December 20, 1988)