Tracheary Element Differentiation Research Articles

Redifferentiation of Single Mesophyll Cells into Tracheary Elements

Plant cells possess a potency to redifferentiate to other types of cells in response to external stimuli. The differentiation of mesophyll cells into tracheary elements (TEs) is an excellent example of redifferentiation occurring at the cellular level in vascular plants. In situ, vessels and tracheids are formed from TEs, which are differentiated from cells of the procambium of the root and shoot in primary xylem or from cells produced by the vascular cambium in secondary xylem (Torrey et al. 1971; Aloni 1987). In vitro, TEs can be induced from various types of cells, including phloem parenchyma cells of carrot roots (Mizuno et al. 1971), cortical cells of pea roots (Phillips and Torrey 1973), pith parenchyma cells of lettuce (Dalessandro and Roberts 1971), tuber parenchyma cells of Jerusalem artichoke (Minocha and Halperin 1974; Phillips and Dodds 1977), mesophyll cells of Zinnia (Kohlenbach and Schmidt 1975; Fukuda and Komamine 1980a), and epidermal cells of Zinnia leaves (Church and Galston 1989), by wounding and/or phytohormone application. TEs are characterized by the formation of a secondary cell wall with annular, spiral, reticulate, or pitted wall thickenings. At maturity, differentiating TEs lose their nuclei and cell contents, forming a hollow tube. The easy induction of differentiation and detection of differentiated cells by their morphological features offer a great advantage for the study of redifferentiation in vascular plants. Fukuda and Komamine (1 980a) established an in vitro experimental system in which single mesophyll cells of Zinnia can redifferentiate directly into TEs without cell division, based on the work of Kohlenbach and Schmidt (1975). The Zinnia system has been considered efficient for the study of redifferentiation into TEs, mainly because of high frequency, high synchrony, and other merits (see Appendix). Physiological, biochemical, and molecular markers have been found in Zinnia by which physiological stages of redifferentiation are defined. Such findings have contributed to recent progress in the study of TE differentiation (Fukuda 1989a, 1992b; Sugiyama and Komamine 1990; Fukuda et al. 1993). Therefore, I will review here recent work on redifferentiation of Zinnia mesophyll cells into TEs and discuss the regulation of redifferentiation.

The Tracheid-Differentiation Factor of Conifer Needles

Under experimental conditions, mature living conifer needles on isolated stem segments induce cells at the junction of the needle trace and the cambium to differentiate into tracheary elements (TEs). The response is an all-or-none event in terms of secondary-wall deposition, but the extent of deposition varies between cells, and hence may serve as a model for developmental plasticity at the cellular level. Attempts to reproduce needle-induced TE differentiation using plant growth regulators such as auxin and cytokinin with or without carbohydrates have been unsuccessful but nevertheless have provided evidence for interactions between the needle-produced tracheid-differentiation factor (tdf) and phytohormones in the regulation of cellular differentiation. Treatment of needle-pair stumps with D-myoinositol 1,4,5-trisphosphate, the second messenger from bovine brain, has induced the tdf response at the needle tracecambium junction; however, unequivocal characterization of the endogenous tdf remains to be ach...

Spread of beet necrotic yellow vein virus in infected seedlings and plants of sugar beet (Beta vulgaris)

Infection of sugar beet roots by beet necrotic yellow vein virus (BNYVV) was investigated with transmission electron microscopy, immunogold labelling and enzyme linked immuno sorbent assay (ELISA). Here we show that infection of sugar beet roots is very fast, occurring during germination. Seedlings grown directly in infected soil showed higher BNYVV infection than plants transplanted into infected soil after seven days of initial growth in sterilized soil. The earlier the initial infection, the faster was its spread. The study showed that a few differentiated cells of the cortex and of the xylem parenchyma were the preferred sites of viral multiplication. The spread of viral infection was slow through differentiated tissues. Intact virions were frequently found in undifferentiated and mature vessel elements and xylem parenchyma, whereas they were rare in sieve elements. Virus particle number in the differentiating tracheary elements was high, suggesting that infection of the vessel elements preceded their differentiation. This would explain increased infection after early inoculation. Even the xylem tissue of the primary root was highly infected, the seedlings lacked virus particles in their hypocotyls and leaves.

Molecular Cloning and Characterization of cDNAs Associated with Tracheary Element Differentiation in Cultured Zinnia Cells

Mesophyll cells isolated mechanically from leaves of Zinnia elegans L. cv Canary bird differentiate into tracheary elements (TE) semisynchronously and at high frequency. Using this system, three cDNA clones, TED2 to TED4, whose corresponding mRNAs were expressed in a close association with tracheary element differentiation, were isolated by differential screening of a lambda gt11 cDNA library. The library was prepared using poly(A)+ RNA from cells cultured in a TE-induced medium for 48 h prior to morphological changes, including secondary cell-wall thickenings and autolysis. Northern analysis indicated that mRNAs corresponding to the clones were expressed preferentially in cells differentiating into TEs prior to the morphological changes. The expression of the mRNAs was found not to be induced by alpha-naphthaleneacetic acid or benzyladenine solely and not to be associated directly with cell division. Analysis of the nucleotide sequence of TED4 showed that the cDNA contains an open reading frame of 285 bp, encoding a polypeptide comprising 95 amino acid residues with a predicted molecular mass of 10.0 kD. A homology search of the nucleotide and amino acid sequences of TED4 with several data bases revealed a significant similarity to those of the barley aleurone-specific clone B11E, which was isolated as an aleurone-specific cDNA from 20-d postanthesis grain.

Xylem changes in the correlative inhibition of lateral bud growth in flax (Linum usitatissimum L.)

Xylem or tracheary changes at the base of the cotyledonary buds of flax seedlings (Linum usitatissimum L.), released from inhibition by decapitation of the main apex were studied. The differentiation of xylem strands and/or tracheary elements was correlated with the growth in length of the lateral buds, especially 48–72 hr after the removal of the main apex. The xylem strands, connected to the hypocotylary stele or not, and the tracheary elements increased with age within and outside the strands of both non-decapitated and decapitated seedlings. In the latter, the differentiation of these structures, however, occurred much earlier and in greater abundance in the same regions. The early growth in length of lateral buds, 1 or 2 hr after decapitation, was correlated with the early development of tracheary perforations in the xylem strands. The xylary strands with perforated elements are known to be more efficient than those without them. Therefore, it is suggested that the inhibition of lateral-bud growth was due, in fact, to a lack of appropriate tracheary perforations in the bud xylem strands that were connected with the hypocotylary stele of flax seedlings.

Interrelationship between lignin deposition and the activities of peroxidase isoenzymes in differentiating tracheary elements of Zinnia

In a culture system in which single cells isolated from the mesophyll of Zinnia elegans L. differentiate to tracheary elements (TEs), two inhibitors of phenylalanine ammonia-lyase (EC 4.3.1.5), L-α-aminooxy-β-phenylpropionic acid (AOPP) at 10 μM inhibited lignification without reducing the number of TEs formed. These inhibitors caused intracellular changes in peroxidase (EC 1.11.1.7) activities. The inhibitors increased the activity of peroxidases bound to the cell walls and especially the activity of peroxidase bound ionically to the cell walls. In contrast, the activity of extracellular peroxidase decreased. There were five isoenzymes, P1-P5, in the ionically bound peroxidase of cultured Zinnia cells. Among the isoenzymes, P4 and P5 appeared to be specific for TE differentation. Treatment with AOPP and AIP resulted in increases in the activities of P2, P4 and P5 isoenzymes, with the most prominent increase in P5 activity. The addition of lignin precursors, including coniferyl alcohol, to the AOPP-treated cells restored lignification, and suppressed the alteration of peroxidase isoenzyme patterns caused by AOPP. The relationship between the wall-bound peroxidases and lignification during TE differentiation is discussed in the light of these results.

Tracheary element differentiation in Zinnia mesophyll cell cultures

Mechanically isolated mesophyll cells of Zinnia elegans differentiate into tracheary elements (TEs) when cultured in a medium containing adequate auxin and cytokinin. Differentiation in this culture system is relatively synchronous, rapid (occuring within 3 days of cell isolation) and efficient (with up to 65% of the mesophyll cells differentiating into TEs), and does not require prior mitosis. The Zinnia system has been used to investigate (a) cytological and ultrastructural changes occurring during TE differentiation, such as the reorganization of microtubules controlling secondary wall deposition, (b) the influences of calcium and of various plant hormones and antihormones on TE differentiation, and (c) biochemical changes during differentiation, including those occurring during secondary wall deposition, lignification and autolysis. This review summarizes experiments in which the Zinnia system has served as a model for the study of TE differentiation.

Methylxanthines reversibly inhibit tracheary-element differentiation in suspension cultures of Zinnia elegans L.

Tracheary-element (TE) differentiation in suspension-cultured mesophyll cells of Zinnia elegans L. was completely inhibited by caffeine and theophylline only when these methylxanthines were applied at least 8 h prior to the appearance of secondary cell-wall thickenings. In contrast, the calcium-channel blocker nifedipine completely inhibited TE differentiation when applied only 2-3 h prior to the onset of secondary cell-wall deposition. This indicates the involvement of a methylxanthine-inhibitable event in TE differentiation that is distinguishable from an event dependent on influx of extracellular calcium. The correlation between the time of appearance of chlorotetracycline fluorescence (an indicator of sequestered Ca(2+)) and loss of methylxanthine effectiveness indicates that inhibition by methylxanthines may result from release of Ca(2+) from intracellular stores. Methylxanthines with high potencies against adenosine 3' ∶ 5'-cyclic monophosphate (cAMP) phosphodiesterase and adenosine receptors were less effective inhibitors of TE differentiation, indicating that inhibition of differentiation by methylxanthines is independent of cAMP metabolism. The role of cAMP in transduction of the cytokinin signal, which was proposed previously on the basis of stimulation of TE differentiation by theophylline, was investigated using the non-hydrolyzable analog 8-bromo-cAMP. Although 8-bromo-cAMP stimulated differentiation in the absence of inductive concentrations of cytokinin, the non-cyclic analog 8-bromo-AMP was even more effective, indicating that 8-bromo-cAMP behaves as a cytokinin analog, rather than a second messenger, in stimulating TE differentiation.

A simplified medium for in vitro tracheary element differentiation in mesophyll suspension cultures from Zinnia elegans L.

A simplified medium has been developed for the differentiation of tracheary elements in suspension cultures of mesophyll cells of Zinnia elegans L. All inorganic salts contained in media used previously were retained in the simplified medium, but most were reduced in concentration. The only organic supplements required for optimum differentiation were thiamine and nicotinic acid, in addition to the plant growth regulators N6-benzylaminopurine and α-naphthyleneacetic acid, and sucrose as a carbon source. Mannitol, an osmoticum, was necessary for rapid, synchronous differentiation. This simplified medium is particularly suitable for studies of the role of Ca2+ in tracheary element differentiation due to the elimination of myo-inositol, an intermediate in the phosphatidyl inositol signal transduction pathway and reduction in the concentrations of Mg2+ and Mn2+, which block calcium channels. It is also possible to eliminate EDTA from the medium, enabling studies using specific calcium chelators. Additional culture variables for the optimization of differentiation are discussed.

Effects of Auxin Concentration on the Dimensions and Patterns of Tracheary Elements Differentiating in Pith Explants

The optimal concentration of IAA (0.03 mM) for tracheary element differentiation on lettuce pith explants was about ten times greater than the optimal concentration for callus proliferation. Related to this, the mean volume per tracheary element increased with increasing IAA concentration, 18-fold between 0.001 mM and 0.3 mM IAA. At the highest concentrations, some pith cells appeared to differentiate directly into tracheary elements, without cell division, resulting in especially large tracheary elements (...)

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 differentiation of isolated mesophyll cells of Zinnia elegans L. into tracheary elements (TEs) but had no effect on cell division when it was added to the culture medium at a concentration of 3.4 μM. In the presence of uniconazole, none of the cytological events characteristic of the processes of TE differentiation, such as aggregation of actin filaments, bundling of microtubules or localized thickening and lignification of secondary walls, was observed. Uniconazole was effective when it was added to the medium within 36 h after the start of culture. Brassinosteroids (0.2 nM brassinolide or 2 μM homobrassinolide), but not gibberellin A3, counteracted the inhibitory effect of uniconazole on TE differentiation. Brassinosteroids were most effective when they were added to cultures between 24 and 30 h after the start of culture. Exogenously applied brassinosteroids promoted TE differentiation. It is suggested that the synthesis of brassinosteroids is essential for the differentiation of the cells into TEs and that uniconazole inhibits this differentiation through its inhibitory effect on the biosynthesis of brassinosteroids.

Expression of phenylalanine ammonia-lyase gene during tracheary-element differentiation from cultured mesophyll cells ofZinnia elegans L.

Expression of the phenylalanine ammonialyase (PAL) gene during tracheary-element differentiation was studied in mesophyll cell-suspension cultures ofZinnia elegans. Dose-response curves of benzyladenine (BA) and α-naphthaleneacetic acid (NAA) were obtained for the cultures in order to achieve the highest percentage of differentiation. The optimal concentrations of BA and NAA were 0.1 mg·l(-1) and 0.06 mg·l(-1), respectively, which normally stimulated about 40% differentiation by 96 h of culture. The effects of the same ratio but different amounts of BA and NAA on tracheary-element formation have been tested and the results indicate that the absolute amounts of BA and NAA rather than the ratio of them were important for tracheary-element formation in theZinnia cultures. The cells when cultured in the presence of 0.001 mg·l(-1) of BA and 0.06 mg·l(-1) of NAA expanded and divided but did not differentiate. The level of PAL activity, synthesis of PAL protein and the level of PAL mRNA peaked during 72 to 96 h when lignin was actively deposited. This indicated that the PAL gene was temporally and preferentially expressed in association with the lignification during tracheary-element differentiation and thus it can be regarded as a molecular marker for the process.

Tracheary-element differentiation in suspension-cultured cells ofZinnia requires uptake of extracellular Ca2+

Tracheary-element (TE) differentiation in suspension cultures ofZinnia elegans L. mesophyll cells was inhibited by blocking calcium uptake in three ways: 1) reducing the [Ca(2+)] of the culture medium, 2) blocking calcium channels with the non-permeant cation La(3+), and 3) blocking calcium channels with permeant dihydropyridine calcium-channel blockers. Calcium-channel blockers were effective when added at any time between 0 and 48 h after culture initiation; after 48h, calcium sequestration and secondary cell-wall deposition began. In contrast, calmodulin antagonists inhibited TE differentiation when added at the beginning of culture, but not when added after 24h. These results indicate that TE differentiation involves at least two calcium-regulated events: one calmodulin-dependent and occurring shortly after exposure to inductive conditions, and the other calmodulin-independent and occurring just prior to secondary cell-wall deposition.

L‐α‐Aminooxy‐β‐phenylpropionic acid inhibits lignification but not the differentiation to tracheary elements of isolated mesophyll cells of Zinnia elegans

The influence of L‐α‐aminooxy‐β‐phenylpropionic acid (AOPP), an inhibitor of L‐phenylalanine ammonia‐lyase (PAL; EC 4.3.1.5), and thus of lignin formation, on the differentiation of tracheary elements from isolated mesophyll cells of Zinnia elegans L. cv. Canary Bird was investigated. At low concentrations of AOPP (5–25 μ,M) lignification of differentiating cells was almost completely prevented whereas number of differentiating cells, viability of the cells, fresh and dry weights, and the packed cell volume were higher than corresponding parameters in control cultures. At higher concentrations of AOPP (50–75 μM) the formation of tracheary elements was inhibited but division, elongation and expansion of cells were still observed. Cells cultured for 96 h in the presence of 100 μM AOPP were morphologically similar to cells at 12 h of culture, the time at which AOPP was added. At concentrations of AOPP that did not inhibit differentiation, AOPP caused an increase in the amounts of uronic acid and total carbohydrate (per unit volume of cell suspension) in the extracellular polysaccharide fraction and in the total cell wall fraction, although these parameters were not significantly different from control values when expressed on a dry weight basis. AOPP caused the release of polysaccharides which contained xylose into the medium when added before the onset of visible differentiation and the release of polysaccharides which contained glucose when added at the time when the formation of the secondary cell wall thickenings took place. The results indicate that AOPP at low concentrations specifically inhibits the formation of lignin without adversely affecting the synthesis of cell wall polysaccharides, although the proper integration of these compounds into the wall may be disturbed. O‐Benzylhydroxyla‐mine, on the other hand, did not prove to be a useful agent to affect lignin synthesis in differentiating Zinnia cells.

Structural aspects of tracheary element differentiation in suspension cultures of Zinnia elegans

Tracheary elements, the water-conducting cells in plants, are characterized by their reinforced walls that became thickened in localized patterns during differentiation (Fig. 1). The synthesis of this localized wall involves abundant secretion of Golgi vesicles that export preformed matrix polysaccharides and putative proteins involved in cellulose synthesis. Since the cells are not growing, some kind of endocytotic process must also occur. Many researchers have commented on where exocytosis occurs in relation to the thickenings (for example, see), but they based their interpretations on chemical fixation techniques that are not likely to provide reliable information about rapid processes such as vesicle fusion. We have used rapid freezing to more accurately assess patterns of vesicle fusion in tracheary elements. We have also determined the localization of calcium, which is known to regulate vesicle fusion in plant and animal cells.Mesophyll cells were obtained from immature first leaves of Zinnia elegans var. Envy (Park Seed Co., Greenwood, S.C.) and cultured as described previously with the following exceptions: (a) concentration of benzylaminopurine in the culture medium was reduced to 0.2 mg/l and myoinositol was eliminated; and (b) 1.75ml cultures were incubated in 22 x 90mm shell vials with 112rpm rotary shaking. Cells that were actively involved in differentiation were harvested and frozen in solidifying Freon as described previously. Fractures occurred preferentially at the cell/planchet interface, which allowed us to find some excellently-preserved cells in the replicas. Other differentiating cells were incubated for 20-30 min in 10(μM CTC (Sigma), an antibiotic that fluoresces in the presence of membrane-sequestered calcium. They were observed in an Olympus BH-2 microscope equipped for epi-fluorescence (violet filter package and additional Zeiss KP560 barrier filter to block chlorophyll autofluorescence).

Rise in chlorotetracycline fluorescence accompanies tracheary element differentiation in suspension cultures ofZinnia

Developing tracheary elements in suspension cultures ofZinnia elegans fluoresce intensely relative to non-differentiating cells when stained with chlorotetracycline (CTC), a fluorescent chelate probe for membrane associated calcium. This suggests that a change in calcium uptake or subcellular distribution accompanies the onset of tracheary element differentiation. A few cells in early differentiating cultures were brightly fluorescent, but did not have visible cell wall thickenings, suggesting that a rise in sequestered calcium may precede visible differentiation. Diffuse CTC fluorescence in early differentiation most likely results from sequestration of calcium in the endoplasmic reticulum. Late in differentiation, CTC fluorescence becomes punctate in appearance, probably due to loss of plasma membrane integrity occurring at the onset of autolysis.Zinnia suspension culture cells were found to be very sensitive to CTC and low concentrations (10 μM) were used to assure accurate localization of membrane-associated calcium in healthy cells.

Dynamic Organization of the Cytoskeleton during Tracheary-Element Differentiation: (actin filament/cytoskeleton/microtubule/tracheary-element differentiation/Zinnia elegans).

Dynamic Organization of the Cytoskeleton during Tracheary-Element Differentiation: (actin filament/cytoskeleton/microtubule/tracheary-element differentiation/Zinnia elegans).

Xylogenesis and hormones in soybean callus. I: A histological and ultrastructural study

Five ultrastructural developmental stages are described for differentiating tracheary elements in callus of Glycine max (L.) Merr. Xylogenesis was induced by exogenous application of indolebutyric acid (IBA) + kinetin, IBA + trans- zeatin, IBA + benzyladenine (BA) and IBA + gibberellic acid (GA 3 ). Initiation of tracheary elements occurred after division of the callus cells and was followed by cell enlargement, secondary wall deposition and lignification, all of which culminated in the final autolysis of the immature tracheary elements. Mature elements included both tracheids with scalariform to reticulate wall sculpturing and pitted vessels with simple perforations. Nodulation was extensive in all the treatments. A lateral pattern in autolysis of tracheary elements is thought to be a reflection of a concomitant migration of the inductive stimulus ultimately resulting in groups of xylem elements. Vyf ultrastrukturele ontwikkelingstadia vir differensiërende trageale elemente in Glycine max (L.) Merr. (soyaboon) kallus word beskryf. Xilogenese is deur die toediening van indoolbottersuur (IBA) + kinetien, IBA + trans- zeatien, IBA + bensieladenien (BA) en IBA + gibberelliensuur (GA 3 ) geïnduseer. Trageale elemente is na seldeling geïnisieer en is gevolg deur selvergroting, sekondêre selwand-deposisie en lignifikasie, wat uiteindelik tot outolise van die ont- wikkelende elemente lei. Volwasse elemente is gekenmerk deur beide trageïede met leervormige tot netvormige selwandstrukture en stippelhoutvate met eenvoudige perforasies. Bondelvorming is volop in al die behandelings. ’n Laterale outolisepatroon in trageale elemente word toegeskryf aan ’n gepaardgaande migrasie van die induktiewe stimulus om uiteindelik in groepies xileemelemente te eindig.

Interrelation between the spatial disposition of actin filaments and microtubules during the differentiation of tracheary elements in culturedZinnia cells

Changes in the spatial relationship between actin filaments and microtubules during the differentiation of tracheary elements (TEs) was investigated by a double staining technique in isolatedZinnia mesophyll cells. Before thickening of the secondary wall began to occur, the actin filaments and microtubules were oriented parallel to the long axis of the cell. Reticulate bundles of microtubules and aggregates of actin filaments emerged beneath the plasma membrane almost simultaneously, immediately before the start of the deposition of the secondary wall. The aggregates of actin filaments were observed exclusively between the microtubule bundles. Subsequently, the aggregates of actin filaments extended preferentially in the direction transverse to the long axis of the cell, and the arrays of bundles of microtubules which were still present between the aggregates of actin filaments became transversely aligned. The deposition of the secondary walls then took place along the transversely aligned bundles of microtubules.

Hormonal induction and antihormonal inhibition of tracheary element differentiation in Zinnia cell cultures

Mechanically isolated mesophyll cells of Zinnia elegans L. cv. Envy differentiate to tracheary elements when cultured in inductive medium containing sufficient auxin and cytokinin. Tracheary element differentiation was induced by the three auxins (α-naphthaleneacetic acid, indole-3-acetic acid, and 2,4-dichlorophenoxyacetic acid) and four cytokinins (6-benzyladenine, kinetin, 2-isopentenyladenine and zeatin) tested. Tracheary element formation is inhibited or delayed if the inductive medium is supplemented with an anticytokinin, antiauxin, or inhibitor of auxin transport.