Transient transformation and RNA silencing in Zinnia tracheary element differentiating cell cultures

The Zinnia elegans cell culture system is a robust and physiologically relevant in vitro system for the study of xylem formation. Freshly isolated mesophyll cells of Zinnia can be hormonally induced to semisynchronously transdifferentiate into tracheary elements (TEs). Although the system has proven to be valuable, its utility is diminished by the lack of an efficient transformation protocol. We herein present a novel method to introduce DNA/RNA efficiently into Zinnia cells by electroporation-based transient transformation. Using reporter gene plasmids, we optimized the system for efficiency of transformation and ability for the transformed cells to transdifferentiate into TEs. Optimal conditions included a partial digestion of the cell walls by pectolyase, a low voltage and high capacitance electrical pulse and an optimal medium to maintain cell viability during transformation. Beyond the simple expression of a reporter protein in Zinnia cells, we extended our protocol to subcellular protein targeting, simultaneous co-expression of several reporter proteins and promoter-activity monitoring during TE differentiation. Most importantly, we tested the system for double-stranded RNA (dsRNA)-induced RNA silencing. By introducing in vitro -synthesized dsRNAs, we were able to phenocopy the Arabidopsis cellulose synthase (CesA) mutants that had defects in secondary cell-wall synthesis. Suppressing the expression of Zinnia CesA homologues resulted in an increase of abnormal TEs with aberrant secondary walls. Our electroporation-based transient transformation protocol provides the suite of tools long required for functional analysis and developmental studies at single cell levels.     

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.     

Flow cytometric analysis of tracheary element differentiation in Zinnia elegans cells.

BACKGROUND: Tracheary element (TE) differentiation in single cells in culture isolated from Zinnia elegans leaves involves programmed cell death (PCD) co-ordinated with key morphological developments. We have used flow cytometry to analyze physiological and nuclear changes in the differentiating cells. Flow cytometry allows the identification of subpopulations, thereby removing the obscuring effect of population heterogeneity that occurs with the use of other techniques. METHODS: Cell viability, plasma membrane integrity, oxidative activity, intracellular calcium and pH, cell wall thickening, the possible role of microtubule rearrangement, chromatin condensation, and DNA breakdown were followed by flow cytometry from the first stages of TE induction. RESULTS: TE differentiation could be enhanced and made more synchronous by a centrifugation step at 72 h after cell isolation. Size and shape changes were the first changes identified in differentiating cells, and these properties could be used to isolate differentiating populations by back-gating. Chromatin condensation and nDNA breakdown followed patterns characteristic of programmed cell death. CONCLUSIONS: We have used flow cytometry to characterize the morphological and physiological changes that occur during TE differentiation, and our findings indicate that this process is a form of autophagic PCD in which microtubule rearrangement appears to play a role.     

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 -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.Abbreviation TE tracheary element     

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.     

Establishing in vitro Zinnia elegans cell suspension culture with high tracheary element differentiation

The Zinnia elegans mesophyll cell culture is a useful system for xylogenesis studies. The system is associated with highly synchronous tracheary element (TE) differentiation, making it more suitable for molecular studies requiring larger amounts of molecular isolates, such as mRNA and proteins and for studying cellulose synthesis. There is, however, the problem of non-uniformity and significant variations in the yields of TEs (%TE). One possible cause for this variability in the %TE could be the lack of a standardized experimental protocol in various research laboratories for establishing the Zinnia culture. Mesophyll cells isolated from the first true leaves of Z. elegans var Envy seedlings of approximately 14 days old were cultured in vitro and differentiated into TEs. The xylogenic culture medium was supplied with 1 mg/l each of benzylaminopurine (BA) and α-naphthalene acetic acid (NAA). Application of this improved culture method resulted in stable and reproducible amounts of TE as high as 76% in the Zinnia culture. The increase was mainly due to conditioning of the mesophyll cell culture and adjustments of the phytohormonal balance in the cultures. Also, certain biochemical and cytological methods have been shown to reliably monitor progress of TE differentiation. We conclude that, with the adoption of current improvement in the xylogenic Z. elegans culture, higher amounts of tracheary elements can be produced. This successful outcome raises the potential of the Zinnia system as a suitable model for cellulose and xylogenesis research.     

Galactoglucomannans increase cell population density and alter the protoxylem/metaxylem tracheary element ratio in xylogenic cultures of Zinnia

Xylogenic cultures of zinnia (Zinnia elegans) provide a unique opportunity to study signaling pathways of tracheary element (TE) differentiation. In vitro TEs differentiate into either protoxylem (PX)-like TEs characterized by annular/helical secondary wall thickening or metaxylem (MX)-like TEs with reticulate/scalariform/pitted thickening. The factors that determine these different cell fates are largely unknown. We show here that supplementing zinnia cultures with exogenous galactoglucomannan oligosaccharides (GGMOs) derived from spruce (Picea abies) xylem had two major effects: an increase in cell population density and a decrease in the ratio of PX to MX TEs. In an attempt to link these two effects, the consequence of the plane of cell division on PX-MX differentiation was assessed. Although GGMOs did not affect the plane of cell division per se, they significantly increased the proportion of longitudinally divided cells differentiating into MX. To test the biological significance of these findings, we have determined the presence of mannan-containing oligosaccharides in zinnia cultures in vitro. Immunoblot assays indicated that beta-1,4-mannosyl epitopes accumulate specifically in TE-inductive media. These epitopes were homogeneously distributed within the thickened secondary walls of TEs when the primary cell wall was weakly labeled. Using polysaccharide analysis carbohydrate gel electrophoresis, glucomannans were specifically detected in cell walls of differentiating zinnia cultures. Finally, zinnia macroarrays probed with cDNAs from cells cultured in the presence or absence of GGMOs indicated that significantly more genes were down-regulated rather than up-regulated by GGMOs. This study constitutes a major step in the elucidation of signaling mechanisms of PX- and MX-specific genetic programs in zinnia.     

Involvement of phosphoinositide turnover in tracheary element differentiation in Zinnia elegans L. cells

Mesophyll cells of Zinnia elegans L., cultured in the presence of phytohormones, will transdifferentiate and undergo programmed cell death to become tracheary elements, thick-walled cells of the xylem. This system is a model system for study of plant cell development and differentiation. We report that a high concentration of extracellular Ca(2+) is necessary during the first 6 h of culturing for tracheary elements to form. Extracellular Ca(2+) is still required at later times, but at a much lower concentration. When cells transdifferentiate in adequate Ca(2+), microsomal phospholipase C activity increases and levels of inositol 1,4,5-trisphosphate rise at about hour 4 of culturing. The production of inositol 1,4,5-trisphosphate appears to be important for tracheary element formation, since inhibitors of phospholipase C inhibit both inositol 1,4,5-trisphosphate production and tracheary element formation. Pertussis toxin, an inhibitor of GTP-binding proteins, inhibits transdifferentiation and eliminates inositol 1,4,5-trisphosphate production. Tracheary element formation was not completely abolished by inhibitors that eliminated inositol 1,4,5-trisphosphate production, suggesting the involvement of other pathways in regulating transdifferentiation.     

Relationship between tracheary element differentiation and the cell cycle in single cells isolated from the mesophyll of Zinnia elegans

A relationship between tracheary element differentiation and the cell cycle was studied in single cells isolated from the mesophyll of Zinnia elegans L. cv. Canary bird. Almost all nuclei of isolated mesophyll cells were at the 2 C level of DNA, indicating that almost all cells were initially in the G₁ phase and that somatic polyploidy was absent. Cultured cells underwent partially synchronous DNA replication at 42 h and mitosis at 54 h of culture, and the first cell cycle time was approximately 58 h.
The occurrence and timing of DNA replication and mitosis during cytodifferentiation to tracheary elements were investigated using microspectrophotometry, microfluorometry, tritiated thymidine autoradiography, and serial observation. More than 55% of the nuclei of the immature tracheary elements were at the 2 C level of DNA and were not labeled by continuous feeding with tritiated thymidine, providing clear evidence that these cells differentiated without interventing DNA replication. Some tracheary elements (approximately 30%) were formed after one round of the cell cycle, and others (less than 5%) were formed after passing through the S phase, but without intervening mitosis. All types of tracheary elements appeared simultaneously after 58 h of culture, and their patterns of increase in number were similar. From the results, we propose a hypothesis concerning the relationship between cytodifferentiation and the cell cycle.     

Involvement of extracellular dilignols in lignification during tracheary element differentiation of isolated Zinnia mesophyll cells

During differentiation of isolated Zinnia mesophyll cells into tracheary elements (TEs), lignification on TEs progresses by supply of monolignols not only from TEs themselves but also from surrounding xylem parenchyma-like cells through the culture medium. However, how lignin polymerizes from the secreted monolignols has not been resolved. In this study, we analyzed phenol compounds in culture medium with reversed-phase HPLC, gas chromatography-mass spectrometry and nuclear magnetic resonance spectrometry, and found 12 phenolic compounds including coniferyl alcohol and four dilignols, i.e. erythro-guaiacylglycerol-beta-coniferyl ether, threo-guaiacylglycerol-beta-coniferyl ether, dehydrodiconiferyl alcohol and pinoresinol, in the medium in which TEs were developing. Coniferyl alcohol applied to TE-inductive cultures during TE formation rapidly disappeared from the medium, and caused a sudden increase in dilignols. Addition of the dilignols promoted lignification of TEs in which monolignol biosynthesis was blocked by an inhibitor of phenylalanine anmmonia-lyase (PAL), L-alpha-aminooxy-beta-phenylpropionic acid (AOPP). These results suggested that dilignols can act as intermediates of lignin polymerization.     

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.     

Separation and characterization of the isoenzymes of wall-bound peroxidase from cultured Zinnia cells during tracheary element differentiation

Cell wall-bound peroxidase (EC 1.11.1.7) isoenzymes (P1-P5) from cells of Zinnia elegans L. that were differentiating into tracheary elements were separated and characterized to obtain information about the relationships between these isoenzymes and the biosynthesis of lignin. Fractionation of Zinnia cells by centrifugation in solutions of Percoll revealed that P1, P2, and P5 were present in differentiated tracheary elements. These peroxidase isoenzymes were separated by several column-chromatographic steps. During hydrophobic chromatography on Phenyl Superose, P5 activity was separated into activities P5A and P5B. Enzymatically pure preparations of P1, P3, P5A, and P5B were finally obtained and used for the characterization of each isoenzyme. The optimum pH was 5.5–6.0 for P1, 5.0–7.5 for P3, 5.0 for P5A, and 4.0 for P5B. Each of the isoenzymes oxidized coniferyl alcohol efficiently, whereas p-coumaryl alcohol and sinapyl alcohol were poor substrates for all the isoenzymes. An absolute requirement for Ca²⁺ ions was demonstrated for P3. Based on these results, possible roles of peroxidase isoenzymes in the formation of lignin during the differentiation of tracheary elements are discussed.Abbreviations DAB diaminobenzidine - GTA equal proportions of 3,3-dimethylglutaric acid, tris(hydroxymethyl)aminomethane, and 2-amino-2-methyl-1,3-propanediol - TE tracheary element The authors are very grateful to Professor M. Tanahashi of Gifu University for providing hydroxycinnamyl alcohols. This work was supported in part by Grants-in-Aid from the Ministry of Education, Science and Culture of Japan to H.F.     

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

Summary 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 M) were used to assure accurate localization of membrane-associated calcium in healthy cells.Abbreviations CTC chlorotetracycline - DIC differential interference contrast - DiOC₆ 3,3-dihexyloxacarbocyanine iodide - ER endoplasmic reticulum - EGTA ethylene glycol bis-(amino-ethyl ether) N,N,N¹N¹-tetraacetic acid - NPN n-phenylnaphthylamine - OsFeCN osmium tetroxide and potassium ferricyanide - TE tracheary element - TEM transmission electron microscopy     

Progress of lignification mediated by intercellular transportation of monolignols during tracheary element differentiation of isolated Zinnia mesophyll cells

Tracheary element (TE) differentiation is a typical example of programmed cell death (PCD) in higher plants, and maturation of TEs is completed by degradation of all cell contents. However, lignification of TEs progresses even after PCD. We investigated how and whence monolignols are supplied to TEs which have undergone PCD during differentiation of isolated Zinnia mesophyll cells into TEs. Higher densities of cell culture induced greater lignification of TEs. Whereas the continuous exchanging of culture medium suppressed lignification of TEs, further addition of coniferyl alcohol into the exchanging medium reduced the suppression of lignification. Analysis of the culture medium by HPLC and GC-MS showed that coniferyl alcohol, coniferaldehyde, and sinapyl alcohol accumulated in TE inductive culture. The concentration of coniferyl alcohol peaked at the beginning of secondary wall thickening, decreased rapidly during secondary wall thickening, then increased again. These results indicated that lignification on TEs progresses by supply of monolignols from not only TEs themselves but also surrounding xylem parenchyma-like cells through medium in vitro.     

Competitive action between Brassinosteroid and tracheary element differentiation inhibitory factor in controlling xylem cell differentiation

For permanent secondary growth in plants, cell proliferation and differentiation should be strictly controlled in the vascular meristem consisting of (pro)cambial cells. A peptide hormone tracheary element differentiation inhibitory factor (TDIF) functions to inhibit xylem differentiation, while a plant hormone brassinosteroid (BR) promotes xylem differentiation in (pro)cambial cells. However, it remains unclear how TDIF and BR cooperate to regulate xylem differentiation for the proper maintenance of the vascular meristem. In this study, I developed an easy evaluation method for xylem differentiation frequency in a vascular induction system Vascular cell Induction culture System Using Arabidopsis Leaves (VISUAL) by utilizing a xylem-specific luciferase reporter line. In this quantitative system, TDIF suppressed and BR promoted xylem differentiation in a dose-dependent manner, respectively. Moreover, simultaneous treatment of TDIF and BR with (pro)cambial cells revealed that they can cancel their each other’s effect on xylem differentiation, suggesting a competitive relationship between TDIF and BR. Thus, mutual inhibition of “ON” and “OFF” signal enables the fine-tuned regulation of xylem differentiation in the vascular meristem.     

A possible role of glutathione and glutathione disulfide in tracheary element differentiation in the cultured mesophyll cells of Zinnia elegans.

We investigated the relationship between the cellular redox state of GSH or GSSG and tracheary element (TE) differentiation using a Zinnia experimental system, in which isolated mesophyll cells transdifferentiate to TEs. TE differentiation was suppressed by the application of L-buthionine sulfoximine (BSO), a potent inhibitor of GSH biosynthesis, at the early stage of cell culture. Application of GSSG at the early culture stage promoted the differentiation, but that of GSH or GSSG at an advanced period of culture suppressed the differentiation. Application of GSH and GSSG nullified the TE differentiation-suppressing effect of BSO. The results suggest that changes in the redox states of GSH and GSSG have a role in TE differentiation.     

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.     

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.