Regulation of Tracheary Element Differentiation

   apd: 7 June 2001     

Determination of Tracheary Element Differentiation in Lettuce Pith Explants

Transfer experiments with lettuce pith explants revealed thatprovision of inductive concentrations of both cytokinin andauxin in the culture medium for the first 3 and 5 d respectivelywas sufficient to cause determination of tracheary elementsas shown by their subsequent differentiation in substantialnumbers in explants removed from the inductive hormonal stimuli. Determination, differentiation, tissue culture, IAA, zeatin, tracheary elements, lettuce, Lactuca     

Loss of Tonoplast Integrity Programmed in Tracheary Element Differentiation

A tracheary element (TE) is a typical example of a cell type that undergoes programmed cell death in the developmental processes of vascular plants. The loss of the selective permeability of the tonoplast, which corresponds to tonoplast disintegration, occurred after the cells commenced secondary wall thickening and played a pivotal role in the programmed cell death of TEs in a zinnia (Zinnia elegans L.) cell culture. A search for events specifically associated with the TE vacuole provided an important clue to the understanding of the cell death mechanism. The transport of fluorescein, a fluorescent organic anion, across the tonoplast declined drastically in differentiating TEs. The capacity of the vacuole to accumulate the probe was also impaired. Treatment with probenecid, an inhibitor of organic anion transport, caused rapid cell death of TEs and led to the ultimate disruption of the vacuole even in other types of cultured cells. These changes in vacuolar properties during TE development were suppressed by cycloheximide. Specific mRNA accumulation in cells cultured in a TE differentiation-inductive condition was abolished by probenecid. These results suggest that a change in vacuolar membrane permeability promotes programmed cell death in TEs.     

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.     

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。     

Regulation of Tracheary Element Differentiation by Exogenous L-Methionine in Callus of Soya Bean Cultivars

The relationship between the induction of tracheary elementdifferentiation and exogenous L-methionine was examined in agar-growncultures of soya bean callus initiated from Glycine max L. ‘Wayne’and ‘Clark 63’. Although Wayne is a normal cultivarsoya bean, seedlings of Clark 63 exhibit abnormal growth at25 °C due to exessive ethylene biosynthesis at this temperature.Wayne callus showed increased xylogenesis in the presence ofexogenous L-methionine (3.7 µg 1^–1) in comparisonto IAA–KN controls at both 20 and 25 °C. Clark 63callus produced greater numbers of tracheary elements in responseto exogenous L-methionine only at 25 °C. The induction ofxylem differentiation was independent of the maintenance temperatureof the stock cultures of both cultivars. Xylogenesis initiatedbyan IAA–KN medium was inhibited by the addition of AgNO₃(20 mg 1^–1) to the extent of 76.5 per cent in cv. Wayneand 6 per cent in cv. Clark 63. The inhibitory effect was partiallyreversed by the addition of L-methionine (3.7 µg 1^–1)to the IAA–KN–AgNO₂ medium. These data support thehypothesis that xylogenesis in vitro involves auxin, cytokininand ethylene. differentiation, xylogenesis, L-methionine, ethylene, Glycine max L., soya bean, callus culture, auxin, kinetin     

Characteristics of the Inhibition of Induced Tracheary Element Differentiation by 3-Aminobenzamide and Related Compounds

3-Aminobenzamide (3-ABAM), a specific inhibitor of the nuclearenzyme ADP-ribosyl transferase (ADP-RT) has been previouslyshown to inhibit the differentiation of tracheary elements withoutaffecting cell division (Hawkins and Phillips, 1983). This effectis confirmed here in cultured explants of pea as well as Jerusalemartichoke, and total inhibition of direct differentiation incultures of gamma-irradiated immature artichoke tubers demonstrated.In studies on the timing of action of 3-ABAM, cells were foundto be sensitive only during the 6 h period prior to the onsetof visible differentiation, i.e., after the final mitosis whencells were known to be in the G₁/G₀ phase of the cell cycle.The duration of effectiveness of 3-ABAM was limited to a maximumof 3–4 d, apparently due to metabolic degradation of theinhibitor. Other reported ADP-RT inhibitors and their analogueswere found to be toxic in this system. The blocking of differentiationby 3-ABAM was freely and rapidly reversed on removal, and bythis means the maturation time for differentiating trachearyelements was estimated at 6 h. The potential of studies on ADP-RTactivity, and its possible role in regulating DNA strand breakageand repair for advancing our understanding of the molecularmechanisms involved in xylem differentiation are discussed. Key words: Tracheary differentiation, 3-Aminobenzamide, Tissue culture     

Tracheary Element Differentiation Induced in Isolated Cylinders of Lettuce Pith: a Bipolar Gradient Technique

To study the influence of morphogenetic gradients on vasculardifferentiation patterns, a new technique was developed whichallows different substances to be applied at opposite ends ofa tissue block. It yielded information on the mobility of particularmorphogens and on the dependence of callus formation and trachearyelement differentiation on their presence. Application of indol-3ylacetic acid (1AA) (10 mg l^–1), zeatin (0.1 mg l^–1)and sucrose (3 per cent, w/v) in various combinations to theends of cylindrical explants of lettuce pith (Lactuca sativaL.) showed that (a) callus formation was stimulated by IAA,whereas induction of tracheary elements required both IAA andzeatin; (b) callus was confined to a few millimetres at theends of the explants, and tracheary elements occurred mainlywithin the callus; (c) sucrose or its metabolic products diffusedthe 10 mm length of the explants, while IAA and zeatin wereeffective only close to the application site; and (d) some callusand tracheary elements formed when no sucrose was applied, butboth increased with sucrose application, though inhibition oftracheary elements formation occurred with high sucrose concentrations. differentiation, pith explant, tissue culture, xylogenesis, indol-3yl acetic acid, sucrose, zeatin, lettuce, Lactuca sativa     

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.     

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     

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.     

Effect of Light and Activated Charcoal on Tracheary Element Differentiation in Callus Cultures of Pinus Radiata D. Don

Light has been found to increase the proportion of tracheary elements differentiating in callus cultures derived from xylem-parenchyma of Pinus radiata D. Don grown on an induction medium containing activated charcoal but no phytohormones. The differentiation rate increased from 20% when callus was grown in darkness to 45% when callus was grown with a 16 h or 24 h photoperiod. When callus was grown with a 16 h photoperiod, tracheary elements were observed 2 days after transfer of callus to the induction medium, as compared to 5 days when callus was cultured in darkness. The differentiation rate was also influenced by the concentration of activated charcoal added to the induction medium, the optimum concentration being 5 g l⁻¹. Exclusion of activated charcoal from the induction medium decreased the differentiation rate to 2%. The activities of the lignin-related enzymes L-phenylalanine ammonia lyase and cinnamyl alcohol dehydrogenase were significantly higher in cell cultures grown with a 16 h photoperiod as compared to when grown in darkness. The results show that light had a stimulating effect on tracheary element differentiation and the activities of lignin-related enzymes in P. radiata callus cultures. The new growth conditions markedly improve this cell culture system and make it particularly useful for functional gene testing and cell-wall analysis of in vitro grown tracheary elements of coniferous gymnosperms.     

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)     

Tracheary Element Differentiation Uses a Novel Mechanism Coordinating Programmed Cell Death and Secondary Cell Wall Synthesis

Tracheary element differentiation requires strict coordination of secondary cell wall synthesis and programmed cell death (PCD) to produce a functional cell corpse. The execution of cell death involves an influx of Ca²⁺ into the cell and is manifested by rapid collapse of the large hydrolytic vacuole and cessation of cytoplasmic streaming. This precise means of effecting cell death is a prerequisite for postmortem developmental events, including autolysis and chromatin degradation. A 40-kD serine protease is secreted during secondary cell wall synthesis, which may be the coordinating factor between secondary cell wall synthesis and PCD. Specific proteolysis of the extracellular matrix is necessary and sufficient to trigger Ca²⁺ influx, vacuole collapse, cell death, and chromatin degradation, suggesting that extracellular proteolysis plays a key regulatory role during PCD. We propose a model in which secondary cell wall synthesis and cell death are coordinated by the concomitant secretion of the 40-kD protease and secondary cell wall precursors. Subsequent cell death is triggered by a critical activity of protease or the arrival of substrate signal precursor corresponding with the completion of a functional secondary cell wall.     

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)     

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)     

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.     

植物导管分子分化和形成的生理生化机制

从木质素代谢、细胞凋亡、生长素在导管分子分化和形成过程中的作用等方面对近年来植物导管分子分化和形成的生理生化机制研究进展作了介绍.