Further Characterization of Expression of Auxin-lnduced Genes in Tobacco (Nicotiana tabacum) Cell-Suspension Cultures (original) (raw)
Related papers
Naturwissenschaften, 2006
Effects of auxin as plant hormones are widespread; in fact in almost all aspects of plant growth and development auxin plays a pivotal role. Although auxin is required for propagating cell division in plant cells, its effect upon cell division is least understood. If auxin is depleted from the culture medium, cultured cells cease to divide. It has been demonstrated in this context that the addition of auxin to auxin-starved nondividing tobacco BY-2 cells induced semisynchronous cell division. On the other hand, there are some cell lines, named habituated cells, that can grow without auxin. The cause and reason for the habituated cells have not been clarified. A habituated cell line named 2B-13 is derived from the tobacco BY-2 cell line, which has been most intensively studied among plant cell lines. When we tried to find the difference between two cell lines of BY-2 and 2B-13 cells, we found that the addition of culture filtrated from the auxin-habituated 2B-13 cells induced semisynchronous cell division in auxin-starved BY-2 cells. The cell division factor (CDF) that is responsible for inducing cell division in auxin-starved BY-2 cells was purified to near-homogeneity by sequential passage through a hydroxyapatite column, a ConA Sepharose column and a Sephadex gel filtration column. The resulting purified fraction appeared as a single band of high molecular weight on sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels by silver staining and was able to induce cell division in auxinstarved BY-2 cells. Identification of the protein by MALD-TOF-MS/MS revealed that it is structurally related to P-glycoprotein from Gossypioides kirkii, which belongs to ATP-binding cassette (ABC)-transporters. The significance of CDF as a possible ABC-transporter is discussed in relationship to auxin-autotrophic growth and auxinsignaling pathway.
Plant Molecular Biology, 1991
In previous studies we have identified several mRNAs which accumulate after addition of 2,4-dichlorophenoxyacetic-acid (2,4-D) to auxin-starved tobacco cells [45, 46]. The mRNAs corresponding to cDNA clone pCNT103 were found to accumulate transiently prior to the cell division response due to auxin treatment. In this study we determined the sequences of three 103-like cDNAs and two 103-like genes, GNT1 and GNT35. To further study the regulation of the expression of these genes their 5' regions were translationally fused with the fl-D-glucuronidase reporter gene (GUS). The GNT1 5' region led to GUS expression only in the root tips of transgenic plants. By using transgenic hairy-root cultures and transformed cell suspension cultures it was shown that the 5' regions of both GNT1 and GNT35 lead to 2,4-D-inducible expression of GUS activity. The homology of the 103-like genes with other auxinregulated genes is evaluated.
The Auxin-induced Growth of Tobacco Callus Tissue
Biochemie und Physiologie der Pflanzen, 1974
The author analysed the steril tissue, isolated from the stem of tobacco (Nicotiana tabacum), grown 34 days on synthetic nutrient medium, supplemented by different amount of auxin. On the basis of data of fresh weight, dry matter, cell number and RNase activity after 34 days and after a short term auxin treatment in relation of RNase activity, it can be stated: 1. The growth of tissue is changed-in dependence with the auxin concentration appliedaccording to an optimum curve. 2. A reverse proportionality was found between the growth and the RNase activity. On the other hand: the short term (2 or 4 hours) auxin treatment caused a remerkable inhibition in activity at the growth-promoting concentrations and vice varsa. The experimental series indicated the probable central role of nucleic acid metabolism and the nucleases in the auxin-action. On the other hand the technique of steril cultivation is a usefu I method in the examinations of the plant growth.
Planta, 1995
The ipt gene from the T-DNA of Agrobacterium tumefaciens was transferred to tobacco (Nicotiana tabacum L.) in order to study the control which auxin appears to exert over levels of cytokinin generated by expression of this gene. The transgenic tissues contained elevated levels of cytokinins, exhibited cytokinin and auxin autonomy and grew as shooty calli on hormonefree media. Addition of 1-naphthylacetic acid to this culture medium reduced the total level of cytokinins by 84% while 6-benzylaminopurine elevated the cytokinin level when added to media containing auxin. The cytokinins in the transgenic tissue were labelled with 3H and auxin was found to promote conversion of zeatin-type cytokinins to 3H-labelled adenine derivatives. When the very rapid metabolism of exogenous [3H]zeatin riboside was suppressed by a phenylurea derivative, a noncompetitive inhibitor of cytokinin oxidase, auxin promoted metabolism to adenine-type compounds. Since these results indicated that auxin promoted cytokinin oxidase activity in the transformed tissue, this enzyme was purified from the tobacco tissue cultures. Auxin did not increase the level of the enzyme per unit tissue protein, but did enhance the activity of the enzyme in vitro and promoted the activity of both glycosylated and non-glycosylated forms. This enhancement could contribute to the decrease in cytokinin level induced by auxin. Studies of cytokinin biosynthesis in the transgenic tissues indicated that transhydroxylation of isopentenyladenine-type cytokinins to yield zeatin-type cytokinins occurred principally at the nucleotide level.
Expression of the isopentenyl transferase gene is regulated by auxin in transgenic tobacco tissues
Transgenic Research, 1996
The ipt gene from the T-DNA of Agrobacterium tumefaciens was transferred to tobacco (Nicotiana tabacum L.) in order to study the control which auxin appears to exert over levels of cytokinin generated by expression of this gene. The transgenic tissues contained elevated levels of cytokinins, exhibited cytokinin and auxin autonomy and grew as shooty calli on hormonefree media. Addition of 1-naphthylacetic acid to this culture medium reduced the total level of cytokinins by 84% while 6-benzylaminopurine elevated the cytokinin level when added to media containing auxin. The cytokinins in the transgenic tissue were labelled with 3H and auxin was found to promote conversion of zeatin-type cytokinins to 3H-labelled adenine derivatives. When the very rapid metabolism of exogenous [3H]zeatin riboside was suppressed by a phenylurea derivative, a noncompetitive inhibitor of cytokinin oxidase, auxin promoted metabolism to adenine-type compounds. Since these results indicated that auxin promoted cytokinin oxidase activity in the transformed tissue, this enzyme was purified from the tobacco tissue cultures. Auxin did not increase the level of the enzyme per unit tissue protein, but did enhance the activity of the enzyme in vitro and promoted the activity of both glycosylated and non-glycosylated forms. This enhancement could contribute to the decrease in cytokinin level induced by auxin. Studies of cytokinin biosynthesis in the transgenic tissues indicated that transhydroxylation of isopentenyladenine-type cytokinins to yield zeatin-type cytokinins occurred principally at the nucleotide level.
Re‐evaluation of phytohormone‐independent division of tobacco protoplast‐derived cells
The Plant …, 1999
published data dealing with the response of tobacco protoplasts to phytohormones, lipochitooligosaccharides and peptides (Harling et al.These proliferation assays reveal that, in contrast to published data, isolated cells of the investigated mutant plant lines axi159 (Hayashi et al., 1992; Walden et al., 1994), axi4/1 (Harling et al., 1997) and cyi1 (Miklashevichs et al., 1997), which were generated by activation T-DNA tagging, were unable to grow in the absence of auxin or cytokinin. Furthermore, lipochitooligosaccharides which play a key role in the induction of nodules on roots of legumes were unable to promote auxin-or cytokinin-independent cell division in tobacco 461 protoplasts as claimed by Rö hrig et al. (1995, 1996). The finding of van de Sande et al. (1996) that ENOD40 confers tolerance of high auxin concentration to wild-type tobacco protoplasts was also reinvestigated. The results of our investigations show that we were unable to reproduce the proliferation data presented in this study, which were obtained by counting tobacco protoplast-derived cells undergoing division. In total, none of the published data on phytohormone-independent division of tobacco cells could be reproduced.
Genes of auxin biosynthesis and auxin-regulated genes controlling plant cell division and extension
xyloglucan-specific α-1,2and α-1,6-fucosyltransferases and glycosyltransferases, CSL genes of xyloglucan glucan synthases and β-1,4-mannan synthases, MUR genes xyloglucan galactosyltransferases as well as the AtXT1 gene and homologous AtGT2-7 genes xyloglucan xylosyltransferases at Arabidopsis; the XS1 gene of xylan synthase at rice, and also GS1 gene of glucan synthase at corn. A possible role of cell wall protein -extensin (encoded by the auxinregulated HRGP gene) in the plants defence from pathogens and unfavourable factors of external environment is discussed .
The role of auxin-binding protein 1 in the expansion of tobacco leaf cells
The Plant Journal, 2002
Tobacco leaf was used to investigate the mechanism of action of auxin-binding protein 1 (ABP1). The distributions of free auxin, ABP1, percentage of leaf nuclei in G2 and the amount of auxin-inducible growth were each determined in control tobacco leaves and leaves over-expressing Arabidopsis ABP1. These parameters were compared with growth of tobacco leaves, measured both spatially and temporally throughout the entire expansion phase. Within a de®ned window of leaf development, juvenile leaf cells that inducibly expressed Arabidopsis ABP1 prematurely advanced nuclei to the G2 phase. The ABP1-induced increase in cell expansion occured before the advance to the G2 phase, indicating that the ABP1-induced G2 phase advance is an indirect effect of cell expansion. The level of ABP1 was highest at the position of maximum cell expansion, maximum auxin-inducible growth and where the free auxin level was the lowest. In contrast, the position of maximum cell division correlated with higher auxin levels and lower ABP1 levels. Consistent with the correlations observed in leaves, tobacco cells (BY-2) in culture displayed two dose-dependent responses to auxin. At a low auxin concentration, cells expanded, while at a relatively higher concentration, cells divided and incorporated [ 3 H]-thymidine. Antisense suppression of ABP1 in these cells dramatically reduced cell expansion with negligible effect on cell division. Taken together, the data suggest that ABP1 acts at a relatively low level of auxin to mediate cell expansion, whereas high auxin levels stimulate cell division via an unidenti®ed receptor.
THE PLANT CELL ONLINE, 1992
Both cytokinin (N6-benzyladenine [BA]) and auxin (2,4-dichlorophenoxyacetic acid [2,4-D]) stimulate the accumulation of an mRNA, represented by the cDNA pLS216, in Nicotiana plumbaginifolia suspension culture cells. The kinetics of RNA accumulation were different for the two hormones; however, the response to both was transient, and the magnitude of the response was dose dependent. Runoff transcription experiments demonstrated that the transient appearance of the RNA could be accounted for by feedback regulation of transcription and not by the induction of an RNA degradation system. The feedback mechanism appeared to desensitize the cells to further exposure of the hormone. In particular, cells became refractory to the subsequent addition of 2,4-D after the initial RNA accumulation response subsided. A very different response was observed when the second hormone was added to cells that had been desensitized to the first hormone. Under such conditions, BA produced a heightened response in cells desensitized to 2,4-D and vice versa. These findings support a model in which cytokinin further enhances the auxin response or prevents its feedback inhibition. The hormone-induced RNA accumulation was blocked by the protein kinase inhibitor staurosporin. On the other hand, the protein phosphatase inhibitor okadaic acid stimulated expression, and, in particular, okadaic acid was able to stimulate RNA accumulation in cells desensitized to auxin. This suggests that hormone activation involves phosphorylation of critical proteins on the hormone signaling pathway, whereas feedback inhibition may involve dephosphorylation of these proteins. The sequence of pLS216 is similar to genes in other plants that are stimulated by multiple agonists such as auxins, elicitors, and heavy metals, and to the gene encoding the stringent starvation protein in Escherichia coli. It is proposed that this gene family in various plants be called multiple stimulus response (msr) genes.
Characterization of two cDNAs encoding auxin-binding proteins in Nicotiana tabacum
Plant molecular biology, 1997
The isolation and the characterization of two tobacco cDNAs, Nt-ERabp1 and Nt-ERabp2, homologous to Zm-ERabp1, encoding the major auxin-binding protein from maize coleoptiles, are described. Their predicted amino acid sequences correspond to proteins of ca. 21 kDa, in which the characteristic regions common to ABP1-related polypeptides are well-conserved. Southern analysis indicates that the genes corresponding to Nt-ERabp1 cDNA and Nt-ERabp2 cDNA derive respectively from Nicotiana tomentosiformis and Nicotiana sylvestris, the diploid progenitors of Nicotiana tabacum. Analysis of mRNA distribution in tobacco plants indicates that these two genes are preferentially expressed in flowers and growing seedlings. Whatever the tissue tested, Nt-ERabp1 mRNA is more abundant than Nt-ERabp2 mRNA. Furthermore, RT-PCR reveals developmental and organ-specific expression of these two genes in flower parts of tobacco plants. In particular, regulation of Nt-ERabp1 mRNA accumulation appears to be co...