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.     

Changes in the activity and mRNA of cinnamyl alcohol dehydrogenase during tracheary element differentiation in zinnia.

Changes in the enzymatic activity of cinnamyl alcohol dehydrogenase (CAD) and in the expression of a gene for CAD during tracheary element (TE) differentiation were investigated in cultures of single cells isolated from the mesophyll of zinnia (Zinnia elegans). In cultures in which TE differentiation was induced (TE-inductive cultures), CAD activity increased from h 36 after the start of culture (12 h before the start of thickening of the secondary cell wall) and peaked at h 72, when lignin was actively being deposited. In control cultures in which TE differentiation was not induced, CAD activity remained at a very low level for 5 d. Some isoforms of CAD were detected only in the TE-inductive cultures by native gel electrophoresis and subsequent staining for CAD activity. A cDNA clone for CAD, ZCAD1, was isolated from Z. elegans using a cDNA clone for CAD from Aralia cordata as the probe. RNA gel-blot analysis revealed that in the TE-inductive cultures the level of ZCAD1 mRNA increased from h 36 and peaked at h 48 to 60. No such increases were observed in control cultures. These results indicated that both the gene expression and the activity of CAD are strictly regulated, in association with lignification, during TE differentiation in Z. elegans.     

Expansion of cultured Zinnia mesophyll cells in response to hormones and light

Mesophyll suspension cultures of Zinnia elegans L. have been used extensively to investigate the development of tracheary elements. Here we have modified the culture conditions to promote cell expansion and inhibit tracheary element differentiation and cell division. Cell expansion, measured by computer image analysis, was stimulated by auxin ( α -naphthyleneacetic acid), cytokinin (N⁶-benzylaminopurine), gibberellic acid, brassinosteroid (24-epibrassinolide), and light, all of which are known to promote cell expansion in whole plants or excised organs. Whereas light stimulated cell expansion primarily during the first 48 h of culture, auxin, cytokinin, gibberellic acid and brassinosteroid had little effect until after 48 h. Treatments also differed in their relative effects on cell elongation and radial cell expansion. Light and cytokinin had a greater effect on radial cell expansion, auxin and epibrassinolide promoted only cell elongation, and gibberellic acid had nearly equal effects on expansion in both directions. We have also shown by combining treatments that the effects of cytokinin and auxin are additive. Neither hormone treatment, however, was additive with the effect of light treatment. Finally, in contrast to xylogenic cultures where expansion occurs by tip growth, cell expansion in non-differentiating cells was due to diffuse growth. These data show that cell expansion can be induced by hormones in primary mesophyll cultures from Zinnia in contrast to serially transferred plant suspension cultures. Furthermore, they indicate that auxin, cytokinin, and light induce cell expansion by different mechanisms in these cultures.     

Expression of extensin genes is dependent on the stage of the cell cycle and cell proliferation in suspension-cultured Catharanthus roseus cells

To isolate cDNAs expressed at a specific phase of the cell cycle in a higher plant, we performed differential screening of a cDNA library prepared from the S-phase cells of synchronized cultures of Catharanthus roseus. Sequence analysis shows that two of the identified cDNAs, cyc15 and cyc17, encode extensins that represent a family of cell wall hydroxyproline-rich glycoproteins. Protein sequences deduced from the two cDNAs contain the characteristic pentapeptide repeat sequence, Ser-Pro-Pro-Pro-Pro, which is commonly observed in extensins. The protein sequences also share several other extensin characteristics such as the presence of a N-terminal signal peptide and a high content of Tyr and Lys residues. When C. roseus cell suspension cultures were synchronized by phosphate starvation, the mRNAs of both cyc15 and cyc17 were transiently expressed during the S and G2 phases of the cell cycle. However, significant amounts of the mRNAs also accumulated in phosphate-starved cells arrested in the G1 phase. In asynchronous cultures, both genes were expressed during the stationary phase, when cell proliferation ceased. The observed patterns of expression suggest that the extensin genes, cyc15 and cyc17, are under two types of regulation: one that depends on the stage of the cell cycle and another that is induced during the growth arrest. Thus, the products of these genes may function both during the progression through the cell cycle and in the strengthening of the cell wall after cell division.     

A fasciclin-domain containing gene, ZeFLA11, is expressed exclusively in xylem elements that have reticulate wall thickenings in the stem vascular system of Zinnia elegans cv Envy

The vascular cylinder of the mature stem of Zinnia elegans cv Envy contains two anatomically distinct sets of vascular bundles, stem bundles and leaf-trace bundles. We isolated a full-length cDNA of ZeFLA11, a fasciclin-domain-containing gene, from a zinnia cDNA library derived from in vitro cultures of mesophyll cells induced to form tracheary elements. Using RNA in situ hybridization, we show that ZeFLA11 is expressed in the differentiating xylem vessels with reticulate type wall thickenings and adjacent parenchyma cells of zinnia stem bundles, but not in the leaf-trace bundles that deposit spiral thickenings. Our results suggest a function for this cell-surface GPI-anchored glycoprotein in secondary wall deposition during differentiation of metaxylem tissue with reticulate vessels.     

Isolation and characterization of differentially expressed genes in the mycelium and fruit body of Tuber borchii

The transition from vegetative mycelium to fruit body in truffles requires differentiation processes which lead to edible fruit bodies (ascomata) consisting of different cell and tissue types. The identification of genes differentially expressed during these developmental processes can contribute greatly to a better understanding of truffle morphogenesis. A cDNA library was constructed from vegetative mycelium RNAs of the white truffle Tuber borchii, and 214 cDNAs were sequenced. Up to 58% of the expressed sequence tags corresponded to known genes. The majority of the identified sequences represented housekeeping proteins, i.e., proteins involved in gene or protein expression, cell wall formation, primary and secondary metabolism, and signaling pathways. We screened 171 arrayed cDNAs by using cDNA probes constructed from mRNAs of vegetative mycelium and ascomata to identify fruit body-regulated genes. Comparisons of signals from vegetative mycelium and fruit bodies bearing 15 or 70% mature spores revealed significant differences in the expression levels for up to 33% of the investigated genes. The expression levels for six highly regulated genes were confirmed by RNA blot analyses. The expression of glutamine synthetase, 5-aminolevulinic acid synthetase, isocitrate lyase, thioredoxin, glucan 1,3-beta-glucosidase, and UDP-glucose:sterol glucosyl transferase was highly up-regulated, suggesting that amino acid biosynthesis, the glyoxylate cycle pathway, and cell wall synthesis are strikingly altered during morphogenesis.     

Inhibition of proteasome activity by the TED4 protein in extracellular space: a novel mechanism for protection of living cells from injury caused by dying cells

In maturation process of tracheary element (TE) differentiation, many hydrolases are activated to execute programmed cell death of TEs. Such hydrolases are released from maturing TEs into extracellular space. The release of hydrolases should be harmful to surrounding cells. The TED4 protein, a tentative plant non-specific lipid transfer protein that is expressed preferentially in TE-induced culture of zinnia (Zinnia elegans L.), is secreted into the apoplastic space prior to and associated with morphological changes of TEs. Our studies on the interrelationship between the TED4 protein and proteolytic activities using an in vitro TE differentiation system of zinnia revealed the following facts. (1) Active proteasome is released into medium at maturation stage of TE differentiation. (2) The TED4 protein forms a complex with proteasome in culture medium. (3) The TED4 protein inhibits proteasome activity in the medium and crude extracts of zinnia cells. (4) The depletion of the TED4 protein from culture medium results in an increase in mortality of other living cells. These results strongly suggest that the secreted TED4 protein acts as an inhibitor of proteasome to protect other cells from undesirable injury due to proteolytic activities exudated from dying TEs.     

Higher extracellular pH suppresses tracheary element differentiation by affecting auxin uptake

In an optimized liquid medium containing auxin and cytokinin, mesophyll cells isolated from Zinnia elegans L. seedlings can be induced to differentiate into tracheary elements (TEs) at high frequency. However, it is known that buffering the medium at neutral pH severely suppresses TE differentiation. In the process of modifying the medium, we found that excessive administration of auxin restored the suppression. Based on this finding, we physiologically characterized auxin actions involved in TE differentiation by focusing on the influence of extracellular pH. First, dose/response relationships between auxin [1-naphthaleneacetic acid (NAA) and 2,4-dichlorophenoxyacetic acid (2,4-D)] concentrations and differentiated cell ratios were determined under various extracellular pH conditions. Secondly, intracellular concentrations of free forms and metabolites of auxin species were determined by analyzing extracts from cells cultured with radiolabeled NAA and 2,4-D under different extracellular pH conditions with liquid scintillation counting and thin-layer chromatography autoradiograms. Higher extracellular pH was found to reduce both the auxin potency for inducing TE differentiation and intracellular auxin accumulation. Reduction levels correlatively varied depending on the auxin species. These results suggest that the weakening in auxin potency at higher extracellular pH is ascribed to lower auxin uptake, which leads to decreased intracellular perception of the auxin signal. A model to predict auxin action that considers membrane transport, metabolism, and the perception of auxin is also presented.     

Isolation and Characterization of Differentially Expressed Genes in the Mycelium and Fruit Body of Tuber borchii

The transition from vegetative mycelium to fruit body in truffles requires differentiation processes which lead to edible fruit bodies (ascomata) consisting of different cell and tissue types. The identification of genes differentially expressed during these developmental processes can contribute greatly to a better understanding of truffle morphogenesis. A cDNA library was constructed from vegetative mycelium RNAs of the white truffle Tuber borchii, and 214 cDNAs were sequenced. Up to 58% of the expressed sequence tags corresponded to known genes. The majority of the identified sequences represented housekeeping proteins, i.e., proteins involved in gene or protein expression, cell wall formation, primary and secondary metabolism, and signaling pathways. We screened 171 arrayed cDNAs by using cDNA probes constructed from mRNAs of vegetative mycelium and ascomata to identify fruit body-regulated genes. Comparisons of signals from vegetative mycelium and fruit bodies bearing 15 or 70% mature spores revealed significant differences in the expression levels for up to 33% of the investigated genes. The expression levels for six highly regulated genes were confirmed by RNA blot analyses. The expression of glutamine synthetase, 5-aminolevulinic acid synthetase, isocitrate lyase, thioredoxin, glucan 1,3-β-glucosidase, and UDP-glucose:sterol glucosyl transferase was highly up-regulated, suggesting that amino acid biosynthesis, the glyoxylate cycle pathway, and cell wall synthesis are strikingly altered during morphogenesis.     

An arabinogalactan protein(s) is a key component of a fraction that mediates local intercellular communication involved in tracheary element differentiation of zinnia mesophyll cells

Local intercellular communication is involved in tracheary element (TE) differentiation of zinnia (Zinnia elegans L.) mesophyll cells and mediated by a proteinous macromolecule, which was designated xylogen. To characterize and isolate xylogen, a bioassay system to monitor the activity of xylogen was developed, in which mesophyll cells were embedded in microbeads of agarose gel at a low (2.0-4.3x10(4) cells ml(-1)) or high density (8.0-9.0x10(4) cells ml(-1)) and microbeads of different cell densities were cultured together in a liquid medium to give a total density of 2.1-2.5x10(4) cells ml(-1). Without any additives, the frequency of TE differentiation was much smaller in the low-density microbeads than in the high-density microbeads. This low level of TE differentiation in the low-density microbeads was attributable to the shortage of xylogen. When cultures were supplemented with conditioned medium (CM) prepared from zinnia cell suspensions undergoing TE differentiation, the frequency of TE differentiation in the low-density microbeads increased remarkably, indicating the activity of xylogen in the CM. The xylogen activity in CM was sensitive to proteinase treatments. Xylogen was bound to galactose-specific lectins such as Ricinus communis agglutinin and peanut agglutinin, and precipitated by beta-glucosyl Yariv reagent. These results indicate that xylogen is a kind of arabinogalactan protein.     

The influence of cytokinin-auxin cross-regulation on cell-fate determination in Arabidopsis thaliana root development

Root growth and development in Arabidopsis thaliana are sustained by a specialised zone termed the meristem, which contains a population of dividing and differentiating cells that are functionally analogous to a stem cell niche in animals. The hormones auxin and cytokinin control meristem size antagonistically. Local accumulation of auxin promotes cell division and the initiation of a lateral root primordium. By contrast, high cytokinin concentrations disrupt the regular pattern of divisions that characterises lateral root development, and promote differentiation. The way in which the hormones interact is controlled by a genetic regulatory network. In this paper, we propose a deterministic mathematical model to describe this network and present model simulations that reproduce the experimentally observed effects of cytokinin on the expression of auxin regulated genes. We show how auxin response genes and auxin efflux transporters may be affected by the presence of cytokinin. We also analyse and compare the responses of the hormones auxin and cytokinin to changes in their supply with the responses obtained by genetic mutations of SHY2, which encodes a protein that plays a key role in balancing cytokinin and auxin regulation of meristem size. We show that although shy2 mutations can qualitatively reproduce the effect of varying auxin and cytokinin supply on their response genes, some elements of the network respond differently to changes in hormonal supply and to genetic mutations, implying a different, general response of the network. We conclude that an analysis based on the ratio between these two hormones may be misleading and that a mathematical model can serve as a useful tool for stimulate further experimental work by predicting the response of the network to changes in hormone levels and to other genetic mutations.     

Analysis of the protein expression profiling during rice callus differentiation under different plant hormone conditions

Plant hormones function to coordinate plant growth and development. While the plant hormones, mainly auxin and cytokinin, are exogenously added to various plant tissue cultures, their effects on the organogenesis are apparent, but little is known concerning the molecular mechanisms by which they function in cultured cells. Rice, as a model plant in monocots, is also suitable to tissue culture studies. Here, we used four types of regeneration mediums with different relative concentrations of cytokinin and auxin for rice callus differentiation, the calli at different differentiation stages were collected for proteomic analysis. 2-dimensional electrophoresis revealed that 213 protein spots significantly differentially expressed during callus differentiation under different hormone conditions. By using mass spectrometry, 183 differentially expressed protein spots were identified to match 157 unique proteins. Most of these differential proteins were cellular/metabolic process-related proteins, whose different expression patterns may be correlated with the cytokinin and auxin regulation. Several hormone-related proteins were prominently featured in differentiated calli as compared with the initiated calli, such as alpha-amylase isoforms, mannose-binding rice lectin, putative dehydration stress-induced protein, cysteine endopeptidase and cystatin. All these results provide a novel insight into how the two plant hormones effect the callus differentiation in rice on the proteomic level.     

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.     

Hormonal control of cell proliferation requires PASTICCINO genes

PASTICCINO (PAS) genes are required for coordinated cell division and differentiation during plant development. In loss-of-function pas mutants, plant aerial tissues showed ectopic cell division that was specifically enhanced by cytokinins, leading to disorganized tumor-like tissue. To determine the role of the PAS genes in controlling cell proliferation, we first analyzed the expression profiles of several genes involved in cell division and meristem function. Differentiated and meristematic cells of the pas mutants were more competent for cell division as illustrated by the ectopic and enlarged expression profiles of CYCLIN-DEPENDENT KINASE A and CYCLIN B1. The expression of meristematic homeobox genes KNOTTED-LIKE IN ARABIDOPSIS (KNAT2, KNAT6), and SHOOT MERISTEMLESS was also increased in pas mutants. Moreover, the loss of meristem function caused by shoot meristemless mutation can be suppressed by pas2. The KNAT2 expression pattern defines an enlarged meristematic zone in pas mutants that can be mimicked in wild type by cytokinin treatment. Cytokinin induction of the primary cytokinin response markers, ARABIDOPSIS RESPONSE REGULATOR (ARR5 and ARR6), was enhanced and lasted longer in pas mutants, suggesting that PAS genes in wild type repress cytokinin responses. The expression of the cytokinin-regulated cyclin D, cyclin D3.1, was nonetheless not modified in pas mutants. However, primary auxin response genes were down-regulated in pas mutants, as shown by a lower auxin induction of IAA4 and IAA1 genes, demonstrating that the auxin response was also modified. Altogether, our results suggest that PAS genes are involved in the hormonal control of cell division and differentiation.     

Hormonal mechanisms for differentiation in plant tissue culture

Summary Cells possess extraordinary powers to organize their molecular processes not only to maintain a cell in a given steady state but also to recognize that state during differentiation. Regulation of these organizational forces appears to be under the control of chemical factors, and a hormonal concept of regulation has evolved. Hormones have been considered to act by reacting with a specific target site. This may be part of their mode of action, but I would like to suggest that a hormone enters and becomes part of a total molecular resonance system. In so doing, the entire molecular system of the cell is modified. Of the known plant hormones, the cytokinins, because of their role in experimentally induced cell division and differentiation, serve as a probe of hormonal involvement in differentiation. Cultured somatic cells of tobacco plants can be induced to undergo differentiation by addition of cytokinin and auxin to the medium. Studies of the cytokinin hormones show a series of diverse molecular involvements. The archetype cytokinin, N⁶-(Δ²-isopentenyl) adenosine (i⁶Ado), occurs in some molecular species of tRNA where it plays a vital role in the codon-anticodon interaction of tRNA and m-RNA. i⁶Ado under-goes extensive metabolism in the tobacco tissue. It is either degraded to adenosine or converted to derivatives that possess biological activity. It is perhaps, therefore, more correct to consider the hormone function as being derived from this total metabolic web. The normal somatic cells of tobacco cultures spontaneously change occasionally into an autonomous form that requires no external growth factors. This line of cells synthesizes i⁶Ado. The metabolic web of the hormone-dependent strain can be perturbed by added auxin but such is not the case in the autonomous strain. These data provide some insight into the altered state of cytokinin activity in which a cell line changes into an autonomous form. Curiously, in become independent of the requirement for exogenous cytokinin, the autonomous tissue becomes sensitive to added cytokinin. i⁶Ado also inhibits the growth of lines of mammalian cancer cells grown in culture. Presented in the formal symposium on Information Transfer in Eukaryotic Cells, at the 26th Annual Meeting of the Tissue Culture Association, Montreal, Quebec, June 2–5, 1975.