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.     

Tracheary element differentiation

Differentiation of tracheary elements (TEs), characterized by formation of a visible secondary cell wall and autolysis, has been regarded as a model system for cytodifferentiation in plants. Fukuda and Komamine (Plant Physiol 65:57–60, 1980a) established an efficient experimental system for TE differentiation from isolated single mesophyll cells of Zinnia elegans. Physiological, cytological, and biochemical analyses of this system have revealed multiple aspects of TE differentiation regarding cell division, DNA synthesis, the cytoskeleton, autolysis, phytohormones, and cell wall components. In addition, molecular biological approaches have identified a number of Zinnia genes associated with TE-specific events. The knowledge obtained in the Zinnia system has been expanded to studies in other plants including Arabidopsis, leading to the identification of key regulators of TE differentiation.     

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.     

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.     

Isolation and characterization of cDNAs encoding xylogenesis-associated and wounding-induced ribonucleases in Zinnia elegans

The study of plant ribonuclease (RNase) functions is complicated by a complex profile of RNase activities detected in tissues. Thus, isolation of individual RNase genes will be desirable for the further understanding of function of each RNase. Here, we describe the isolation of cDNAs encoding two RNases, ZRNaseI and ZRNaseII, in differentiating tracheary elements (TEs) induced from isolated mesophyll cells of Zinnia elegans. Both the ZRNaseI and ZRNaseII exhibit putative secretion signal sequences at the amino-terminal ends with predicted molecular masses of 24 247 Da and 22 448 Da as mature proteins, respectively. DNA gel blot analysis showed that both RNases in Zinnia appear to be encoded by a small gene family. RNA gel blot analysis showed that the expression of the ZRNaseI gene was associated with the late stage of in vitro TE differentiation, whereas the ZRNaseII gene was mainly induced in response to stress. Neither RNase gene was induced in response to phosphate starvation, or to H₂O₂ challenge in the cultured mesophyll cells, or to senescence in the leaves. In young leaves, the ZRNaseI gene was not induced in response to wounding. But the ZRNaseII gene was markedly induced by 6 h after wounding. Tissue print hybridization showed that the expression of the ZRNaseI gene was preferentially associated with the differentiating TEs in Zinnia stems, while the ZRNaseII mRNA was not detected in unwounded Zinnia organs. Taken together, the results indicate that the ZRNaseI gene is expressed during the process of xylogenesis both in vitro and in the plant, whereas the ZRNaseII gene is predominantly induced in response to wounding. The identification of these RNase genes provides molecular tools for the dissection of the process of autolysis during xylogenesis, and for the dissection of the role of RNase in wounding response.Dedicated to Dr Joseph Elmer Varner.     

Induction of cysteine and serine proteases during xylogenesis in Zinnia elegans

The terminal process of xylogenesis, autolysis, is essential for the formation of a tubular system for conduction of water and solutes throughout the whole plant. Several hydrolase types are implicated in autolysis responsible for the breakdown of cytoplasm. Here, we characterize p48h-17 cDNA from in vitro tracheary elements (TEs) of Zinnia elegans which encodes a preproprotein similar to papain. The putative mature protein, a cysteine protease, has a molecular mass of 22,699 Da with a pI of 5.7. DNA gel blot analysis indicated that p48h-17 is likely encoded by one or two genes. The p48h-17 mRNA accumulated markedly in in vitro differentiating TEs, whereas it appeared not to be induced in response to senescence and wounding in the leaves or H₂O₂ challenge in the cultured mesophyll cells. In stems, the expression of the p48h-17 gene was preferentially associated with differentiating xylem. Activity gel assays demonstrated that a cysteine and a serine protease, which had apparent molecular masses of 20 kDa and 60 kDa, respectively, were markedly induced during in vitro TE differentiation. The cysteine protease activity was also preferentially present in the xylem of Zinnia stems. Transient expression of the p48h-17 cDNA in tobacco protoplasts resulted in the production of a 20 kDa cysteine protease. Taken together, the results indicate that the p48h-17 gene appears to be preferentially associated with xylogenesis, and both the cysteine and serine proteases might be involved in autolysis during xylogenesis.     

Galactoglucomannan oligosaccharides are assumed to affect tracheary element formation via interaction with auxin in Zinnia xylogenic cell culture

Key message

Galactoglucomannan oligosaccharides seem to interact with auxin in xylogenic cell culture, thus influencing mainly metaxylem-like tracheary element differentiation depending on timing with hormones and the process kinetics.

Abstract

Complex mapping of Zinnia mesophyll cell transdifferentiation into tracheary elements with or without prior cell division was documented after palisade and spongy parenchyma cell immobilization during the first 4 days of culture. Here, we report a positive effect of galactoglucomannan oligosaccharides on cell viability and density and higher metaxylem-like tracheary element formation in xylogenic cell culture. The maximal positive effect was achieved by the simultaneous addition of the oligosaccharides and growth hormones (auxin, cytokinin) to the cell culture medium. Moreover, a large number of metaxylem-like tracheary elements were observed in a low-auxin medium supplemented with oligosaccharides, but not in a low-cytokinin medium, suggesting a close relationship between auxin and the oligosaccharides during tracheary element formation.     

Chitosan and a fungal elicitor inhibit tracheary element differentiation and promote accumulation of stress lignin-like substance in Zinnia elegans xylogenic culture

We investigated the effect of elicitors on xylem differentiation and lignification using a Zinnia elegans xylogenic culture system. Water-soluble chitosan and a fungal elicitor derived from Botrytis cinerea were used as elicitors. Elicitor addition at the start of culturing inhibited tracheary element (TE) differentiation in a concentration-dependent manner, and 30 μg mL^?1 of chitosan or 16.7 μg mL^?1 of the fungal elicitor strikingly inhibited TE differentiation and lignification. Addition of chitosan (at 50 μg mL^?1) or the fungal elicitor (at 16.7 μg mL^?1) during the culturing period also inhibited TE differentiation without inhibiting cell division, except for immature TEs undergoing secondary wall thickening. Elicitor addition after immature TE appearance also caused the accumulation of an extracellular lignin-like substance. It appears that elicitor addition at the start of culturing inhibits the process by which dedifferentiated cells differentiate into xylem cell precursors. Elicitor addition during culturing also appears to inhibit the transition from xylem cell precursors to immature TEs, and induces xylem cell precursors or xylem parenchyma cells to produce an extracellular stress lignin-like substance.     

Involvement of gibberellin in tracheary element differentiation and lignification in Zinnia elegans xylogenic culture

Summary. Gibberellin (GA) is considered an important growth regulator involved in many aspects of plant development. However, little is known about the relationship between GA and lignification. In this study, we analyzed the role of GA in tracheary element (TE) differentiation and lignification using a Zinnia elegans xylogenic culture. When gibberellic acid-3 (GA₃) was exogenously supplied, a slight increase in the frequency of TE differentiation and a remarkable increase in lignin content were observed. Computer image analysis of individual TEs showed that the lignification level of each TE was significantly increased in the culture treated with GA₃ compared with those of the control. In contrast, suppression of TE differentiation and lignification was observed when GA biosynthesis was inhibited by ancymidol, paclobutrazol, or uniconazole. This suppression was restored by the addition of GA₃. These results suggest that GA plays an important role in TE differentiation, and even more so in lignification. When conditioned medium obtained after 120 h of control culture was analyzed by high-performance liquid chromatography, many lignin precursors were detected. However, these lignin precursors were greatly reduced in the GA-treated culture. This result suggests that GA promotes lignification by activating the polymerization of lignin precursors. Correspondence and reprints: Department of Biology, Faculty of Science, Ehime University, Matsuyama 790-8577, Japan.     

Involvement of local intercellular communication in the differentiation of zinnia mesophyll cells into tracheary elements

The transdifferentiation of isolated mesophyll cells of zinnia (Zinnia elegans L.) into tracheary elements (TEs) has been well studied as a model of plant cell differentiation. In order to investigate intercellular communication in this phenomenon, two types of culture method were developed, in which mesophyll cells were embedded in a thin sheet of agarose gel and cultured on solid medium, or embedded in microbeads of agarose gel and cultured in liquid medium. A statistical analysis of the two-dimensional distribution of TEs in the thin-sheet cultures demonstrated their aggregation. In the microbead cultures, the frequency of TE differentiation was shown to depend on the local cell density (the cell density in each microbead): TE differentiation required local cell densities of more than 10⁵ cells ml⁻¹. These results suggest that TE differentiation involves cell-cell communication mediated by a locally acting diffusible factor. This presumptive factor was characterized by applying a modified version of the sheet culture, which used two sheets of different cell densities, a low-density sheet and a high-density sheet. Differentiation of TEs in the former could be induced only by bringing it into contact with the latter. Insertion of a 25-kDa-cutoff membrane between the high-density and low-density sheets severely suppressed such induction of TEs in the low-density sheet while a 300-kDa-cutoff membrane suppressed induction only slightly. Insertion of agarose sheets containing immobilized pronase E or trypsin also interfered with the induction of TEs in the low-density sheets. Thus, a proteinaceous macromolecule of 25–300 kDa in molecular weight was assumed to mediate the local intercellular communication required for TE differentiation. This substance was designated “xylogen” with reference to its xylogenic activity. The time of requirement for xylogen during TE differentiation was assessed by experiments in which cells in the low-density sheet were separated from xylogen produced in the high-density sheet at various times by insertion of a 25-kDa-cutoff membrane between the two sheets, and was estimated to be from the 36th hour to the 60th hour of culture (12–36 h before visible thickening of secondary cell walls of TEs). Received: 13 July 2000 / Accepted: 4 October 2000     

Programmed cell death of tracheary elements as a paradigm in plants

Plant development involves various programmed cell death (PCD) processes. Among them, cell death occurring during differentiation of procambium into tracheary elements (TEs), which are a major component of vessels or tracheids, has been studied extensively. Recent studies of PCD during TE differentiation mainly using an in vitro differentiation system of Zinnia have revealed that PCD of TEs is a plant-specific one in which the vacuole plays a central role. Furthermore, there are recent findings of several factors that may initiate PCD of TEs and that act at autonomous degradation of cell contents. Herein I summarize the present knowledge about cell death program during TE differentiation as an excellent example of PCD in plants.     

Cytodifferentiation in isolated single cells

Isolated mesophyll cells ofZinnia elegans synchronously differentiate to tracheary elements at a high frequency when cultured in a liquid medium containing naphth-aleneacetic acid and benzyladenine. An experimental system with the isolatedZinnia mesophyll cells has been extensively used as an efficient model system for the study of cytodifferentiation in higher plants, providing many valuable results on cytodifferentiation. In this review, I introduce the results obtained with theZinnia system and discuss the induction and the sequence of differentiation. Recipient of the Botanical Society Award for Young Scientists, 1987.     

Tip growth in xylogenic suspension cultures ofZinnia elegans L.: Implications for the relationship between cell shape and secondary-cell-wall pattern in tracheary elements

Summary The relationship between cell expansion, cortical microtubule orientation, and patterned secondary-cell-wall deposition was investigated in xylogenic cell suspension cultures ofZinnia elegans L. The direction of cell expansion in these cultures is pH dependent; cells elongate at pH 5.5–6.0, but expand isodiametrically at pH 6.5–7.0. Contrary to our expectations, indirect immunofluorescence revealed that cortical microtubules are oriented parallel to the long axis in elongating cells. Pulse labeling of the walls of isolated cells with the fluorochrome Tinopal LPW demonstrated that xylogenic Zinnia mesophyll cells elongate by tip growth in culture. These results confirm that cortical microtubules in developing tracheary elements reorient before bundling to form transverse cortical microtubule bands. This rearrangement may allow the secondary cell wall pattern to conform to cell shape, independent of the direction in which the cell was expanding prior to reorientation.Abbreviations CMT cortical microtubules - Mes 2-[N-morpholino]ethanesulfonic acid - TE tracheary element     

Purification of cationic peroxidases bound ionically to the cell walls from the roots ofZinnia elegans

Cell wall-bound and tracheary element-specific peroxidase isoenzymes, designated P5A and P5B, were shown previously to be associated with lignification during the differentiation into tracheary elements of single cells isolated from the mesophyll ofZinnia elegans (Satoet al. Planta 189: 584–589, 1993; Planta 196: 141–147, 1995). Isoenzymes corresponding to P5 (RP5A and RP5B) were present at a relatively high level in the roots ofZinnia elegans. These isoenzymes were purified from theZinnia roots by several column-chromatographic steps. Both RP5A and RP5B had molecular masses of 35 kDa. Purified RP5A and RP5B were cleaved by CNBr and the partial amino acid sequences of these isoenzymes were determined.     

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.     

Identifying New Components Participating in the Secondary Cell Wall Formation of Vessel Elements in Zinnia and Arabidopsis

Xylem vessel elements are hollow cellular units that assemble end-to-end to form a continuous vessel throughout the plant body; the xylem vessel is strengthened by the xylem elements'' reinforced secondary cell walls (SCWs). This work aims to unravel the contribution of unknown actors in xylem vessel differentiation using the model in vitro cell culture system of Zinnia elegans differentiating cell cultures and the model in vivo system of Arabidopsis thaliana plants. Tracheary Element Differentiation-Related6 (TED6) and TED7 were selected based on an RNA interference (RNAi) screen in the Zinnia system. RNAi reduction of TED6 and 7 delayed tracheary element (TE) differentiation and co-overexpression of TED6 and 7 increased TE differentiation in cultured Zinnia cells. Arabidopsis TED6 and 7 were expressed preferentially in differentiating vessel elements in seedlings. Aberrant SCW formation of root vessel elements was induced by transient RNAi of At TED7 alone and enhanced by inhibition of both TED6 and 7. Protein–protein interactions were demonstrated between TED6 and a subunit of the SCW-related cellulose synthase complex. Our strategy has succeeded in finding two novel components in SCW formation and has opened the door for in-depth analysis of their molecular functions.     

New insights into pioneer root xylem development: evidence obtained from Populus trichocarpa plants grown under field conditions

Background and Aims

Effective programmed xylogenesis is critical to the structural framework of the plant root system and its central role in the acquisition and long-distance transport of water and nutrients. The process of xylem differentiation in pioneer roots under field conditions is poorly understood. In this study it is hypothesized that xylogenesis, an example of developmental programmed cell death (PCD), in the roots of woody plants demonstrates a clearly defined sequence of events resulting in cell death. A comprehensive analysis was therefore undertaken to identify the stages of xylogenesis in pioneer roots from procambial cells to fully functional vessels with lignified cell walls and secondary cell wall thickenings.

Methods

Xylem differentiation was monitored in the pioneer roots of Populus trichocarpa at the cytological level using rhizotrons under field conditions. Detection and localization of the signalling molecule nitric oxide (NO) and hydrogen peroxide (H₂O₂) was undertaken and a detailed examination of nuclear changes during xylogenesis was conducted. In addition, analyses of the expression of genes involved in secondary cell wall synthesis were performed in situ.

Key Results

The primary event in initially differentiating tracheary elements (TEs) was a burst of NO in thin-walled cells, followed by H₂O₂ synthesis and the appearance of TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling)-positive nuclei. The first changes in nuclear structure were observed in the early stages of xylogenesis of pioneer roots, prior to lignification; however, the nucleus was detectable under transmission electron microscopy in differentiating cells until the stage at which vacuole integrity was maintained, indicating that their degradation was slow and prolonged. The subsequent sequence of events involved secondary cell wall formation and autophagy. Potential gene markers from the cinnamyl alcohol dehydrogenase (CAD) gene family that were related to secondary wall synthesis were associated with primary xylogenesis, showing clear expression in cells that undergo differentiation into TEs and in the thin-walled cells adjacent to the xylem pole.

Conclusions

The early events of TE formation during pioneer root development are described, together with the timing of xylogenesis from signalling via NO, through secondary cell wall synthesis and autophagy events that are initiated long before lignification. This is the first work describing experiments conducted in planta on roots under field conditions demonstrating that the process of xylogenesis in vivo might be gradual and complex.     

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.