JXB Advance Access originally published online on September 6, 2006
Journal of Experimental Botany 2006 57(12):3271-3282; doi:10.1093/jxb/erl089
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RESEARCH PAPER |
ENOD40 affects elongation growth in tobacco Bright Yellow-2 cells by alteration of ethylene biosynthesis kinetics
1Laboratory of Molecular Biology, Department of Plant Sciences, Wageningen University, Dreijenlaan 3, 6703 HA, Wageningen, The Netherlands
2Institute of Biology, Leiden University, Wassenaarseweg 64, 2333 AL, Leiden, The Netherlands
To whom correspondence should be addressed. E-mail: Henk.Franssen{at}wur.nl
Received 31 March 2006; Accepted 21 June 2006
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Abstract |
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Plant developmental processes are controlled by co-ordinated action of phytohormones and plant genes encoding components of developmental response pathways. ENOD40 was identified as a candidate for such a plant factor with a regulatory role during nodulation. Although its mode of action is poorly understood, several lines of evidence suggest interaction with phytohormone response pathways. This hypothesis was investigated by analysing cytokinin-, auxin-, and ethylene-induced responses on cell growth and cell division in transgenic 35S:NtENOD40 Bright Yellow-2 (BY-2) tobacco cell suspensions. It was found that cell division frequency is controlled by the balance between cytokinin and auxin in wild-type cells and that this regulation is not affected in 35S:NtENOD40 lines. Elongation growth, on the other hand, is reduced upon overexpression of NtENOD40. Analysis of ethylene homeostasis shows that ethylene accumulation is accelerated in 35S:NtENOD40 lines. ENOD40 action can be counteracted by an ethylene perception blocker, indicating that ethylene is a negative regulator of elongation growth in 35S:NtENOD40 cells, and that the NtENOD40-induced response is mediated by alteration of ethylene biosynthesis kinetics.
Key words: BY-2 cells, elongation growth, ENOD40, ethylene
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Introduction |
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ENOD40 homologues have been identified in plant species across the plant kingdom, including monocots like rice (Kouchi et al., 1999), rye grass, barley (Larsen et al., 2003), Zea mays (Compaan et al., 2003), and sorghum, and dicots such as tomato (Vleghels et al., 2003), tobacco (Matvienko et al., 1996), citrus, and numerous leguminous species. The highest expression levels of ENOD40 have been found during legume nodule formation, and therefore its function has been studied in most detail during this process. Misregulation of ENOD40 in Medicago truncatula by co-suppression reduces the number of nodules and nodule development is arrested, indicating that ENOD40 has a regulatory role in nodule organogenesis (Crespi et al., 1994; Charon et al., 1999). Ectopic expression of ENOD40, on the other hand, induces cortical cell divisions in Medicago roots and accelerates nodule development (Charon et al., 1997, 1999). However, ENOD40 expression alone is not sufficient for nodule primordium formation (Minami et al., 1996; Mathesius et al., 2000), and interaction with other plant factors is probably required for the initiation of nodule development. Several observations (Hirsch et al., 1989; Peters and Crist-Estes, 1989; Lee and LaRue, 1992; Cooper and Long, 1994; Heidstra et al., 1997) show the involvement of phytohormones, in particular auxin, cytokinin, and ethylene, and of ENOD40 (Charon et al., 1997) suggesting that, during nodule development, cross-talk between ENOD40 and phytohormone signalling exists. The expression of ENOD40 homologues in developmental processes in non-leguminous plant species, for example, during lateral root formation, flower development, and vascular tissue development (Kouchi et al., 1999; Varkonyi-Gasic and White, 2002; Vleghels et al., 2003), indicates that the function of ENOD40 is not confined to nodule development in leguminous species and suggests that ENOD40 has a general role in plant development. This notion is supported by the observation that ectopic expression of ENOD40 affected formation of somatic embryos of alfalfa under in vitro culture conditions (Crespi et al., 1994). Also, overexpression of ENOD40 led to reduced apical dominance in tobacco (van de Sande et al., 1996). Both observations indicate that phytohormone signalling is affected by ENOD40. Up to now, the function of ENOD40 and its mode of action have been poorly understood. Although observations in both legumes and non-legumes are pointing to a cross-talk between ENOD40 activity and phytohormone signalling pathways, direct evidence for such an interaction is lacking. Establishing whether the function of ENOD40 involves interaction with phytohormone signalling pathways could be an important step towards unravelling the role of ENOD40 during organogenesis. Therefore, a search was made for a system that would make it possible to test whether cross-talk between ENOD40 and phytohormone signalling occurs. The tobacco Bright Yellow-2 (BY-2) cell suspension was chosen as a model system as it is convenient for studying phytohormone responses on a cellular level. In BY-2 cells, elongation growth and cell division are regulated by the balance between cytokinin and auxin in the culture medium (Hasezawa and Syono, 1983). Thus, cell elongation growth and cell division frequency can be used as morphological markers to study whether overexpression of ENOD40 affects the response of BY-2 cells to phytohormones. It was found that overexpression of ENOD40 negatively affects cell elongation growth, whereas cytokinin- or auxin-dependent control of cell division frequency is not affected in 35S:NtENOD40 transgenic cell lines. It was shown further that the altered ethylene biosynthesis kinetics observed in ENOD40-overexpressing cells is a primary cause of the reduction in cell elongation growth.
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Materials and methods |
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Construction of binary vector p35S:NtENOD40
Nicotiana tabacum contains two ENOD40 homologues that are 96% identical at the nucleotide level (Matvienko et al., 1996). The cauliflower mosaic virus 35S promoter from pMON999 (Monsanto) was transferred to pCambia 1390 (Cambia, Australia) yielding p35S:Tnos. A 470 bp PCR fragment corresponding to the NtENOD40-1 cDNA sequence was then cloned in p35S:Tnos.
Liquid BY-2 cultures and BY-2 transformation
Nicotiana tabacum BY-2 cell suspensions were subcultured weekly by 40x dilution in fresh medium (Nagata et al., 1981). BY-2 transformation was performed using a modification of the procedure reported by Gu and Verma (1997). Five millilitres of a 3-d-old BY-2 cell suspension was co-cultivated for 2 d at 25 °C in the dark with 60 µl of log-phase Agrobacterium tumefaciens strain C58C1, harbouring the binary vector. Cells were washed three times before plating on culture medium supplemented with 0.8% Daishin agar, 200 µg l–1 ticarciline/clavuline, and 40 µg l–1 hygromycin B. Transgenic calli that appeared after 3–4 weeks, were cultured on fresh selection plates for 1 more week, and were subsequently transferred to liquid selection medium. Six independent 35S:NtENOD40 BY-2 cell lines were generated and named lines Nt1 to Nt6. Each transgenic line was derived from a different callus, which means that they cannot be siblings. Transgenic lines were continuously maintained in selection medium.
Protoplast isolation
Protoplasts were obtained from 6-d-old suspension cultures using 1% (w/v) cellulase-YC and 0.1% (w/v) pectolyase Y23 in 0.4 M D-mannitol, pH 5.5 (Nagata et al., 1981). Cells were incubated in the enzyme solution for 3 h at room temperature, filtered through 63 µm nylon mesh, washed twice with 0.2 M KCl, purified over a one-step 18% (w/v) sucrose gradient, and subsequently washed three times with protoplast culture medium (PCM) containing 4.3 g l–1 MS salts (without vitamins) supplemented with 1 mg l–1 thiamine-HCl, 100 mg l–1 myo-inositol, 10 g l–1 sucrose, 255 mg l–1 KH2PO4, and 0.4 M D-mannitol at pH 5.7. Elongation growth-inducing PCM contained 0.1 mg l–1 1-naphthalene-acetic acid (NAA) and 1.0 mg l–1 benzyl-adenine (BA). Protoplasts were cultured in 3 ml liquid medium at a density of approximately 105 ml–1 in small sealed Petri dishes at 25 °C in the dark (Kuss and Cyr, 1992).
Protoplast assay growth parameter measurements
Growth parameter measurements were performed on random photographs of protoplast-derived cells after 4 d of culture. Viable cells were selected for measurements using FDA (fluorescein-diacetate) staining (Fig. 1). Fluorescent images were captured using a cooled CCD camera mounted on a Leica DMR microscope with a x20 objective. The digital fluorescent images facilitated computer-based morphometric measurements using the NIH-IMAGE program (http:/rsb.info.nih.gov/nih-image) in which objects can be contoured by applying the invert/threshold option. The parameters measured were number of cells per file, cell width, and cell file length. For each sample, the average cell division frequency was calculated as (total number of cells/number of cell files)–1. The cell file length was first expressed in width units, by calculating the length:width ratio for individual cells and then averaged over 100–150 cells per sample. Elongation growth was calculated as increase in cell file length during the culture time as cell file lengthend–cell file lengthbegin, where cell file lengthbegin=1 for a spherical protoplast. Values presented in the graphs represent average values and standard deviations for each line/condition, calculated over a number of independent repetitions, as indicated in the text.
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In order to determine elongation growth capacity of the six individual transgenic lines, protoplasts from the wild-type and the Nt1–Nt6 cell lines were cultured for 4 d in PCM in the presence of 1.0 mg l–1 BA and 0.1 mg l–1 NAA. At least five independent experiments were performed. Pairwise comparisons between transgenic lines and the wild type were performed using a two-tailed Student's t test. Significance values were adjusted for multiple tests.
RNA gel blot analysis
Total RNA was isolated using the TRIzol method (GibcoBRL). A 16 µg aliquot of total RNA was subjected to electrophoresis on a 1% agarose gel in 0.01 M NaH2PO4 (pH 7.0) using the glyoxal/DMSO method. RNA was subsequently transferred to a genescreen membrane in 20x SSC. RNA gel blots were hybridized with radiolabelled PCR fragments of the respective transcripts in formamide hybridization buffer overnight at 42 °C. Autoradiograms were obtained using a Molecular Dynamics phosphorimager (Sunnyvale, CA, USA).
Reverse transcriptase-mediated PCR
Total RNA was isolated using the TRIzol method (GibcoBRL). After DNase I (Promega) treatment to remove chromosomal DNA, cDNA is synthesized from 2.5 µg of total RNA in a volume of 20 µl [10 mM TRIS-HCl pH 8.8, 50 mM KCl, 5 mM MgCl2, 1 mM dNTPs, 1 µg oligo-dT(12)V anchor primer, 20 U RNA guard (Pharmacia), and 200 U MuMLV reverse transcriptase (RT; Stratagene)]. The samples were incubated for 1 h at 37 °C and subsequently at 95 °C for 5 min to inactivate the enzyme. The samples were then diluted to 100 µl and 1 or 2 µl of the cDNA were used for PCR analysis [10 mM TRIS-HCl pH 8.3, 50 mM KCl, 2.5 mM MgCl2, 100 µM dNTPs, 50 ng primer, and 1 U Taq polymerase (Boehringer Mannheim, USA) in a total volume of 50 µl].
Primer sets were designed for RT-mediated PCR-based transcript quantification for each of the genes analysed. Specificities of the primer sets were verified by sequencing the RT-PCR products. The number of PCR cycles was adapted to the linear range of the PCR amplification reaction for each gene, corresponding to the relative expression levels. All samples were normalized on ubiquitin levels. The following primers were used for RT-PCR: UBI-f, 5'-ATGCAGAT(C/T)TTTGTGAAGAC-3'; UBI-r, 5'-ACCACCACG(G/A)AGACGGAG-3'; ACS-f, 5'-GATTTAATACAAGAATGGG-3'; ACS-r, 5'-GAACAATGAAAAGAACAAC-3'; ACO-f, 5'-GGGCTTCTTTGAGTTGGTG-3'; ACO-r, 5'-CTCCGCTGCCTCTTTCTC-3'. Amplified DNA fragments were run on a 1% agarose gel, alkaline blotted to Hybond-N+ membrane (Amersham Pharmacia) and hybridized to radiolabelled PCR fragments of the corresponding cloned cDNAs. Autoradiograms were obtained by using a Molecular Dynamics phosphorimager.
RACE-PCR on NtENOD40 transcripts was as follows: cDNA was synthesized from RNA isolated from the transgenic lines, using the RACE-T anchor primer 5'-CATCTAGAGGATCGAATTC-T(16)-3'. The PCR cycles were 94 °C for 5 min; 30 cycles of 94 °C for 20 s, 50 °C for 20 s, 72 °C for 30 s, and a final extension at 72 °C for 5 min using, in the first run, primers RACE-A, 5'-CATCTAGAGGATCGAATTC-3', and reverse primer, 5'-CGGGATCCTAGTTGGAGTGAATTAAGGA-3', and, in the second run, RACE-A primer and reverse primer, 5'-AAGCTTTTGGAGTCTTTCTTGGCCTTT-3'. After the second PCR, the total RACE-PCR product mixture was purified using a PCR purification kit (Boehringer) and was cloned in pGEM-T (Promega).
Dose–response curves
Protoplasts from three lines (WT, Nt1, and Nt2) were cultured for 4 d in PCM with various concentrations of cytokinin (0.0 mg l–1 BA; 0.1 mg l–1 BA; 0.5 mg l–1 BA; 1.0 mg l–1 BA; and 2.0 mg l–1 BA) and a fixed concentration of auxin (0.1 mg l–1 NAA), or with various concentrations of auxin (0.0 mg l–1 NAA; 0.05 mg l–1 NAA; 0.1 mg l–1 NAA; 0.5 mg l–1 NAA; and 1.0 mg l–1 NAA) and a fixed concentration of cytokinin (1.0 mg l–1 BA). The auxin/cytokinin dose–response curve (DRC) experiment was repeated six, three, and five times for the wild-type, Nt1, and Nt2 lines, respectively. Significant line (PL), dosage (PD), and the interaction between line and dosage (PLxD) effects were obtained by two-way analysis of variance (ANOVA) using the SAS glm procedure (windows version 9.1; SAS Inc, North Carolina, USA). The model applied was: yijk=µ+Li+Dj+LxDij+
ijk, where yijk is either the elongation growth or the cell division frequency from line i (i=1, 2, 3), dosage j (j=1, ..., 5), and observation k (k=1, ..., 6). µ represents the overall mean, L is the main effect for lines, D is the concentration or dosage effect for BA or NAA, LxD is the interaction effect, and is the stochastic error. Differences of the least square means (LSMeans) for the linexdosage effects, along with associated t-tests and P-values, were calculated. For the DRC experiment of the wild type, single factor analysis was performed to estimate the significance of the dosage effect, using the model yjk=µ+Dj+jk. Duncan's multiple range test was applied to compare the effects of the different concentrations. In a similar set-up, the ethylene perception blocker AgNO3 was applied at a concentration range from 10–8 M to 10–5 M with 10-fold increments to protoplasts of wild-type, Nt1, and Nt2 lines, cultured in PCM supplemented with 0.1 mg l–1 NAA and 1.0 mg l–1 BA. To give the appropriate final concentrations of AgNO3, 30 µl of a serial dilution of AgNO3 in water was transferred to the culture medium containing the protoplasts just before sealing the Petri dishes at the start of the culture period. Three independent sets of experiments were performed. Regression analysis of each line was performed using SPSS 9.0 (SPSS, Chicago, IL, USA). The slopes (means ± standard error) of the linear functions obtained for the lines Nt1, Nt2, and wild type were 54.500±24.000 (P=0.041; R2=0.28); 90.500 ±13.000 (P <0.001; R2=0.80), and –2.790±28.300 (P=0.923; R2=0.004), respectively.
Ethylene measurements
For each line, protoplasts were divided over six Petri dishes at the start of the experiment and were cultured in parallel. Each Petri dish was sampled every 24 h for 7 d. In order not to severely alter accumulating ethylene levels, gas samples of 1 ml from a total of 30 ml headspace volume, were taken with a syringe through a rubber gasket in the lid of the Petri dish without opening the sealed Petri dishes. Ethylene concentration was determined directly by standard GC-analysis on a gas chromatograph equipped with an alumina column and a flame ionization detector (Gilissen and Hoekstra, 1984). Ethylene accumulation at each time-point was determined as the average ethylene concentration in the headspace of these six cultures.
Distribution of materials
Upon request, all novel materials described in this publication will be made available in a timely manner for non-commercial research purposes.
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Results |
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Cytokinin–auxin DRCs of BY-2 cells
To create a reference to determine the effect of NtENOD40 overexpression on the phytohormone response of BY-2 cells, cytokinin and auxin DRCs of wild-type BY-2 cells were made by measuring elongation growth and cell division as a function of increasing cytokinin or auxin concentration. It has been shown previously that the most accurate data concerning cytokinin- and auxin-regulated elongation growth and cell division in BY-2 cells are obtained with a bioassay starting from BY-2 protoplasts, which subsequently divide and elongate (Hasezawa and Syono, 1983). The reason for using protoplasts in these assays is that the composition of the BY-2 cell suspension is heterogeneous with respect to cell file length and number of cells per file. By preparing protoplasts from the cell suspension, a population of single cells with a similar diameter is obtained. Analysing growth parameters of these cultured protoplasts has an advantage over using the cell suspension directly as it allows the effects on elongation growth and cell division to be separated. Firstly, under the conditions of the present study the number of cell files during culture remains similar to the number of protoplasts at the start of the experiment. So cells remain attached to each other after division. Hence, by starting from protoplasts, the increase in the number of cells per cell file directly reflects the number of cell divisions that took place during the incubation time, and this parameter is from here onwards called the ‘cell division frequency’. This parameter is expressed as the average number of cells per cell file–1, i.e. cultured protoplasts remain single cells when no cell division takes place, whereas finding two cells per file means that one round of cell division has occurred during the incubation time. Secondly, the width of cells remains similar during culture to the diameter of protoplasts at the start of the incubation (Fig. 1A, B). This means that no radial expansion growth occurs and the length of the cell files at the end of the culture period is a measure of elongation growth. This parameter is hereafter called the ‘cell file length’ and is expressed in width units. Elongation growth is calculated as the increase in cell file length during the culture time. During the incubation time of 4 d used in these bioassays, wild-type cells can become, on average, about four to five times as long as a protoplast. So, the increase in cell length is about three to four times the initial size of a protoplast.
DRCs were made using protoplasts prepared from the wild-type cell line (see Materials and methods). For the cytokinin DRC, elongation growth and cell division frequency were determined as a function of cytokinin (BA) concentration at a fixed concentration of auxin (0.1 mg l–1 NAA). For the auxin DRC, the same parameters were determined as a function of auxin (NAA) concentration at a fixed concentration of cytokinin (1.0 mg l–1 BA) (see Materials and methods). Although data for the wild-type line were obtained in parallel with that of the transgenic lines (see below), the results for the wild-type line are discussed first to illustrate the action of cytokinin and auxin in wild-type BY-2 cells. The DRCs were made six times, in independent experiments, for the wild-type line. The results from independent experiments were similar and the average value (±standard deviation) was calculated for each parameter (Fig. 2A, B). The cytokinin DRC for elongation growth (Fig. 2A) shows that elongation growth is maximal when cells are grown in the absence of exogenous cytokinin. Application of increasing concentrations of cytokinin gradually reduces cell file length of wild-type cells, suggesting that addition of cytokinin has a mild negative effect on elongation growth (one-way ANOVA, PD=0.0985). Application of 2 mg l–1 BA can provoke a reduction of cell file length of 0.78 units (corresponding to a 21% reduction of elongation growth) compared with the cell file length reached when cells are grown in the absence of exogenous cytokinin. Duncan's multiple range test indicated that this difference is significant at the 0.05 level. Thus, exogenous cytokinin is not essential for elongation growth when auxin is present and exogenous application of cytokinin has a mild negative effect on elongation growth. The auxin DRC for cell file length (Fig. 2B) shows that cell file length is not affected (one-way ANOVA, PD=0.8991) by the concentration of auxin as, in the absence of auxin, it is similar to that at the various concentrations of exogenous auxin. This shows that elongation growth neither requires auxin, nor does auxin markedly affect it, when cytokinin is applied to the medium. These data show that the presence of either cytokinin or auxin is sufficient to sustain the growth rates achieved under the culture conditions of the present study and that only addition of cytokinin has a mild negative effect on elongation growth.
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The cytokinin DRC for cell division frequency (Fig. 2A) shows that the cell division frequency is reduced from 0.78 to 0.12 at increasing concentrations of cytokinin, and that the cell division frequency is highest in the absence of exogenous cytokinin. The auxin DRC for cell division frequency (Fig. 2B) shows that the cell division frequency increases from 0.00 to 0.64 at increasing concentrations of auxin. One-way ANOVA analysis indicated that the cell division responses provoked by cytokinin and auxin are highly significant (PD <0.0001). In the absence of auxin, all cell files still consist of a single cell after 4 d of culture, which means that cells have not divided during the culture period. These results show that exogenously applied auxin is essential for cell division in BY-2 cells. Taken together, these observations show that exogenously applied cytokinin has an inhibitory effect on cell division and that exogenous auxin has a stimulating effect on cell division, whereas elongation growth is only mildly reduced by exogenous application of cytokinin in wild-type BY-2 cells.
Generation of stably transformed BY-2 cell lines carrying 35S:NtENOD40
To determine whether overexpression of NtENOD40 affects responses to phytohormones in BY-2 cells, a set of six independent 35S:NtENOD40 BY-2 cell lines, called lines Nt1 to Nt6, was generated by Agrobacterium-mediated transformation (see Materials and methods). Expression of the transgenes was determined by RNA gel blot analysis (Fig. 3A). In the wild-type line, NtENOD40 mRNA could not be detected, indicating a very low expression level of the endogenous NtENOD40 gene. In the six 35S:NtENOD40 lines, NtENOD40 transcripts were expressed at various levels. In all lines except line Nt6, this level was much higher than in the wild-type line, with the highest expression in lines Nt1 and Nt2 and the lowest expression in Nt5 and Nt6. Further, hybridization with the NtENOD40 probe resulted in two bands on the RNA gel blot. To characterize the nature of these two RNAs further, 3'-RACE-PCR was performed on NtENOD40 transcripts of the transgenic lines. Analysis of nucleotide sequences of 11 cloned RACE-PCR fragments revealed that all sequences are fully identical to the transgene sequence and that read-through occurs at the NOS terminator that flanks the NtENOD40 cDNA sequence in the construct, resulting in transcripts of two different lengths (data not shown).
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Cytokinin–auxin DRCs of 35S:NtENOD40 transgenic BY-2 cells
To select lines in which the effect of NtENOD40 overexpression on hormone responses can be studied, first the effect of ENOD40 overexpression was determined on elongation growth and division frequency of cells grown in the presence of 1.0 mg l–1 BA and 0.1 mg l–1 NAA. These conditions represent the intersection of the two sets of conditions used in the DRCs (Fig. 2). To this end, protoplasts were obtained from the wild-type and Nt1–Nt6 cell lines and subsequently cultured for 4 d in PCM (see Materials and methods). Representative photographs taken directly after protoplast isolation, as well as after 4 d of culture of wild-type cells and cells of a transgenic line with a strong phenotype (line Nt1) are presented in Fig. 1A–C. These pictures show the typical elongated appearance of wild-type cells after 4 d of culture, whereas cells are markedly smaller in line Nt1. Wild-type and transgenic cells are of similar size during propagation of the cell suspension culture and so the size of isolated protoplasts is equal. Cell file length and cell division frequency were determined for each line in at least five independent experiments (see Materials and methods). The results from independent experiments were similar and the average value (±standard deviation) was calculated for each parameter (Fig. 3B, C). It was found that four lines, Nt1, Nt2, Nt3, and Nt4, have a strongly reduced cell file length as compared with the wild type (Fig. 3B). Elongation growth in these lines ranged from 1.66 units (Nt1) to 0.84 units (Nt3), corresponding to 56% and 29%, respectively, of the elongation growth of the wild-type line, and is statistically significant (two-tailed Student's t-test, P <0.001) for these four lines. The elongation growth of lines Nt5 and Nt6 is similar to the wild type (Fig. 3B). The cell division frequency in the wild-type line is 0.15±0.09. This means that about 15% of the cells have divided once. The cell division frequency in the transgenic lines is similar to that of the wild type (Fig. 3C). This shows that division frequency is not affected in the transgenic lines. Taken together, these data show that the lines with the highest expression level (Nt1 and Nt2) display the strongest phenotype and that in the lines with the lowest level of NtENOD40 expression no phenotypical change is observed. This indicates that the observed suppression of elongation growth most likely is correlated to overexpression of NtENOD40.
To analyse the effect of NtENOD40 overexpression on cell division frequency and elongation growth in more detail and to test for a possible effect of ENOD40 on auxin or cytokinin responses, DRCs were made for the transgenic lines Nt1 and Nt2, the two lines that have the highest level of NtENOD40 expression and a strong phenotype, and are compared with those of the wild-type line (Fig. 4A–D). As in the wild-type line, the cell division frequency in both transgenic lines shows a strong dose-dependent response to both BA and NAA. The division frequency drops from 0.42 to 0.15 for Nt1 cells and from 0.70 to 0.18 for Nt2 cells grown in the presence of 0.1 mg l–1 NAA at increasing concentrations of BA. For the wild type the division frequency decreases from 0.78 to 0.12 under these conditions. In the presence of 1.0 mg l–1 BA and at increasing concentrations of NAA the division frequency increases from 0.00 to 0.30 for Nt1 cells and from 0.00 to 0.40 for Nt2 cells, while for the wild type the division frequency increases from 0.00 to 0.64. Two-way ANOVA analysis indicated a highly significant dosage effect for BA (PD <0.0001) as well as for NAA (PD <0.0001) in both transgenic lines. Although division frequencies of line Nt1, but not of line Nt2, appear slightly lower than that of the wild type, they are only significantly different between line Nt1 and the wild type under conditions that most strongly induce cell division (at 1.0 mg l–1 BA and 1.0 mg l–1 NAA, P=0.0012; at 0.1 mg l–1 NAA and 0 mg l–1 BA, P=0.0104). Thus, these results indicate that both the inhibitory effect of cytokinin (Fig. 4A) and the stimulating effect of auxin (Fig. 4B) on cell division are similar in wild-type and Nt1 and Nt2 lines and that overexpression of ENOD40 does not alter the response to these hormones.
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Next, the effect of ENOD40 overexpression on elongation growth was examined in the cytokinin–auxin DRCs (Fig. 4C, D). In all conditions tested, a strong reduction of elongation growth in Nt1 and Nt2 cells was observed when compared with that of wild-type cells cultured in the same respective conditions. This reduction of elongation growth is observed throughout both DRCs, and ranges from at least 1.86 units and 1.66 units (50% and 45% of elongation growth of corresponding wild-type cells), respectively, for Nt1 and Nt2 cells cultured at 0 mg l–1 BA and 0.1 mg l–1 NAA, to a maximal 1.96 units (Nt1) and 2.24 units (Nt2) (66% and 76%, respectively), for cells cultured at 1.0 mg l–1 BA and 0 mg l–1 NAA. These data reveal that suppression of elongation growth in 35S:NtENOD40 does not require exogenous cytokinin when cells are cultured in the presence of 0.1 mg l–1 NAA, nor does it require exogenous auxin when 1.0 mg l–1 BA is applied. Two-way ANOVA analysis indicated that elongation growth of Nt1 as well as Nt2 cells differs significantly (PL <0.0001) from that of wild-type cells throughout both DRCs.
Next, it was investigated whether, in addition to the strong negative effect of ENOD40 overexpression on elongation growth, cytokinin or auxin affect elongation growth in the transgenic lines. Two-way ANOVA analysis revealed a significant line effect (PL <0.0001), but did not show a significant auxin-dose effect (PD=0.3038), indicating that auxin does not affect elongation growth in any of the lines, nor did it show a significant interaction between line and dosage effects (PLxD=0.9421), indicating that the response to exogenous auxin is not different between the three lines.
By contrast, application of increasing concentrations of cytokinin gradually reduces cell file length of both transgenic lines (Fig. 4C), suggesting that addition of cytokinin has a mild negative effect on elongation growth, similar to that observed in the wild-type line. (However, two-way ANOVA with PD <0.0001 gave P-values for the differences of the LSMeans of the linexdosage effects which were only slightly significant between the different cytokinin concentrations within one line.) So overexpression of ENOD40 and exogenous cytokinin both negatively affect elongation growth in a similar manner. Two-way ANOVA analysis revealed no significant interaction effects between cytokinin dosage and line (PLxD=0.9968), indicating that overexpression of ENOD40 does not significantly affect the response to exogenous cytokinin. Therefore, it seems unlikely that cytokinin and ENOD40 interact in elongation growth reduction.
ENOD40 affects ethylene homeostasis
It has previously been shown that ethylene is involved in the regulation of root and hypocotyl growth. Analysis of the triple response during ethylene treatments or in mutants of ethylene synthesis or perception pathways has revealed that ethylene can act as a negative regulator of elongation growth in Arabidopsis seedlings (Kieber et al., 1993; Le et al., 2001). Based on this and on the observation that elongation growth is strongly reduced in NtENOD40-overexpressing BY-2 cells, it was tested whether ethylene could be involved in the mechanism that alters the elongation growth response in 35S:NtENOD40 lines. The effect was compared of an ethylene perception blocker on cell division and elongation growth in the lines Nt1 and Nt2, and the wild-type line. Thus, AgNO3 was applied during culture of protoplasts in PCM supplemented with 1.0 mg l–1 BA and 0.1 mg l–1 NAA. In three independent experiments, cell file length and cell division frequency were scored after 4 d of culture. The results from independent experiments were similar and the average value (±standard deviation) was calculated for each parameter (Fig. 5A, B). The results show that cell file length of wild-type cells is similar in the absence and presence of a range of AgNO3 concentrations (Fig. 5A). Thus, a block of ethylene perception has no significant effect (one-way ANOVA, PD=0.9420) on elongation growth in wild-type cells. In the absence of ethylene perception blockers, cell file length of the transgenic lines is reduced by 1.47 units (Nt1) and 1.64 units (Nt2) compared with that of the wild type, in agreement with previous experiments (Figs 3B, 4C). This suppression of elongation growth corresponds to 56% (Nt1) and 62% (Nt2) of elongation growth of the wild-type cells cultured in the absence of AgNO3. By application of 10 µM AgNO3 at the start of the protoplast culture, cell file length of the lines Nt1 and Nt2 is only reduced by 0.92 units (34%) and 0.68 units (25%), respectively, compared with that of the wild type (Fig. 5A). A representative photograph taken after 4 d of culture of cells of line Nt1 in the presence of 10 µM AgNO3 is presented in Fig. 1D. This shows the elongated appearance of these Nt1 cells, indicating that a block of ethylene perception restores the growth defect caused by overexpression of NtENOD40. Two-way ANOVA analysis revealed a significant (PLxD=0.019) interaction between line and dosage, indicating that the differential response of these lines to AgNO3 depends on the dosage. Indeed, further investigation by regression analysis showed a positive linear relationship between AgNO3 dosage and elongation growth for both transgenic lines (Nt1 Pregr=0.041; Nt2 Pregr<0.001), whereas this is not the case for the wild-type line (Pregr=0.932). As in the wild type, the division frequency of the transgenic cells is, at all concentrations of AgNO3 tested, similar to the division frequency in the absence of AgNO3 (Fig. 5B). Two-way ANOVA analysis indicated neither a significant dose effect (PD=0.5383) nor a line effect (PL=0.8239), indicating that neither AgNO3, nor NtENOD40 overexpression affects the division frequency. Taken together, these observations show that an ethylene perception blocker counteracts ENOD40 action and, therefore, indicate that the suppressing effect of ENOD40 on elongation growth is, at least in part, mediated by ethylene.
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Ethylene-mediated responses can be regulated at the level of ethylene production and/or sensitivity. To discriminate between these two possibilities, ethylene production kinetics of wild-type, Nt1, and Nt2 lines cultured in the presence of 1.0 mg l–1 BA and 0.1 mg l–1 NAA were compared. The headspace of protoplast cultures was sampled at 24 h intervals for 7 d and ethylene concentrations were determined by gas chromatography (see Materials and methods). The experiment was performed five times with similar results. One representative experiment is presented in Fig. 6A. In the wild-type culture, ethylene gradually accumulates up to 6 d of culture to a level of about 2 µl l–1. After this time, production ceases. In cultures of the Nt1 and Nt2 lines, ethylene accumulates to similar maximal levels as in wild-type cultures, but transgenic lines already reached a maximal level (2 µl l–1) around day 3. These results show that ethylene production is accelerated in 35S:NtENOD40 lines while the maximal levels are similar in wild-type and Nt1 and Nt2 lines.
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Ethylene accumulation is regulated by ACS but not ACO expression
The next step was to find out how ethylene biosynthesis is accelerated in Nt1 and Nt2 lines. Ethylene biosynthesis can be regulated at different levels, including transcriptional control of gene expression and post-translational regulation of ACC synthase (ACS) and ACC oxidase (ACO) (Wang et al., 2002). Therefore, expression kinetics of these genes in wild-type and transgenic cell lines were analysed until day 4, the typical time-point for scoring growth parameters in these studies. In order to compare ACS and ACO expression levels and ethylene accumulation during culture of protoplasts, protoplasts were cultured in the presence of 0.1 mg l–1 NAA and 1.0 mg l–1 BA and cells of each line (wild type, Nt1, and Nt2) were harvested on days 0, 2, and 4 for RNA extraction. RT-PCR-based ACS and ACO transcript quantification was performed using primers targeted to highly conserved sequences, such that most likely all ACS, respectively ACO, transcripts that are expressed in BY-2 cells can be amplified in a single RT-PCR reaction (see Materials and methods).
ACS and ACO transcript expression profiles (Fig. 6B) are correlated to a time-course of ethylene accumulation (Fig. 6A). In wild-type cultures, ACS transcripts gradually accumulate during the 4 d culture period, and maximal ACS expression levels are found on day 4. By contrast, in 35S:NtENOD40 lines the maximal ACS transcript level is found on day 0, directly after protoplast isolation, and gradually decreases during the culture period (Fig. 6B). These results show that, in 35S:NtENOD40 lines, ACS transcripts accumulate at an earlier time-point, and this is consistent with the accelerated ethylene production. In wild-type cultures, as well as in 35S:NtENOD40 cultures, ACS expression profiles correlate with the timing of ethylene production. In wild-type cultures, ACO transcripts are present directly after protoplast culture has started, and their level only slightly increases during the 4 d culture period. The ACO transcript accumulation profiles in both 35S:NtENOD40 lines are similar to that in the wild type (Fig. 6B). The temporal regulation of ACO transcript accumulation does not correlate with the timing of ethylene production in the different lines. Since a tight correlation between ethylene biosynthesis and ACS, but not ACO, transcript accumulation is found, regulation of ethylene biosynthesis can be largely attributed to transcriptional regulation of ACS.
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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In this report, the interaction of ENOD40 with hormone signalling pathways was studied by analysing hormone-induced responses in tobacco BY-2 cell lines. For this purpose, the BY-2 protoplast assay described by Hasezawa and Syono (1983) was adapted and the dependence of cell elongation growth and cell division on cytokinin, auxin, and ethylene in wild-type and transgenic 35S:NtENOD40 BY-2 cells was investigated.
It was found, in agreement with other studies in BY-2 cells (Hasezawa and Syono, 1983; Hasezawa et al., 1988; Nagata et al., 1992), that cell division frequency is controlled by the cytokinin:auxin ratio. Exogenous cytokinin has an inhibitory effect, whereas auxin stimulates cell division (Fig. 2). The response to different cytokinin or auxin concentrations is comparable in wild-type and 35S:NtENOD40 lines (Fig. 4), suggesting that ENOD40 overexpression in BY-2 cells does not affect cytokinin- or auxin-dependent control on cell division. In addition, evidence is shown that, under the conditions of the present study, alterations of ethylene production or perception do not affect cell division frequency. Similar cell division frequencies are observed in wild-type and transgenic lines (Figs 3–5), despite the differences in their ethylene production levels during early days of protoplast culture (Fig. 6). Also, no effect of ethylene perception blockers on cell division frequency was found (Fig. 5). Therefore it is concluded that ethylene is not involved in the control of cell division under the conditions of the present study.
Elongation growth is not affected by exogenous auxin (Fig. 2), nor is this process sensitive to application of ethylene perception blockers in wild-type BY-2 cells (Fig. 5). Thus, elongation growth does not seem to be limited by either of these two hormones in wild-type cells. By contrast, cytokinin has a mild negative effect as addition of cytokinin reduces elongation growth of wild-type cells in a dose-dependent manner (Fig. 2).
Upon overexpression of NtENOD40, elongation growth is strongly reduced (Figs 3, 4). Analysis of ethylene homeostasis showed that ENOD40-provoked phenotypic responses are mediated by ethylene. Ethylene biosynthesis is accelerated in 35S:NtENOD40 lines, under conditions where ENOD40 reduces elongation growth. Constitutive expression of ENOD40 does not lead to constitutive ethylene production, but rather accelerates a transient accumulation of ethylene during cell growth (Fig. 6). In addition, it was observed that the ENOD40-induced response is counteracted by an ethylene perception blocker (Fig. 5), but this recovery appears not to be complete. This could imply that the complete block of ethylene perception requires a higher AgNO3 concentration that might be toxic to BY-2 cells or that ethylene is not the only factor that is responsible for reduced elongation growth in 35S:NtENOD40 cells. As cytokinin also negatively affects elongation growth, an effect of NtENOD40 overexpression on the endogenous cytokinin concentration cannot be excluded as a factor by which overexpression of NtENOD40 affects elongation growth. However, an increase in the endogenous cytokinin concentration is expected to suppress elongation growth (Fig. 4A) and cell division (Fig. 4C) simultaneously. Since cell division frequencies are not significantly different between wild-type and transgenic lines, it appears unlikely that endogenous cytokinin concentrations are markedly affected by overexpression of NtENOD40. Hence, if besides ethylene a second factor is responsible for reduced elongation growth, the nature of this factor remains unknown.
Nevertheless, the observations made in the present study that ENOD40-provoked responses are mediated by ethylene and that these responses can be counteracted by addition of an ethylene perception blocker, show that acceleration of ethylene accumulation is a primary cause rather than a consequence of the reduced cell elongation in the transgenic lines. Therefore, it seems likely that altered regulation of ethylene biosynthesis is part of the mechanism that affects elongation growth in NtENOD40-overexpressing BY-2 cells. As accelerated accumulation of ACS transcript levels is correlated with accelerated ethylene accumulation in ENOD40-overexpressing BY-2 cells (Fig. 6), ACS could be a primary inducer of the ethylene accumulation. This would be consistent with ACS as the rate-limiting step of ethylene biosynthesis (Yang and Hoffman, 1984) and a well-known primary target of ethylene biosynthesis regulation (Yang and Hoffman, 1984; Theologis, 1992). However, since ethylene accumulation is measured indirectly by analysing the gas phase above the cell culture it cannot be excluded that the cells have been exposed to a higher ethylene concentration before ACS expression is up-regulated. Since ethylene can induce ACS expression, in the latter case, ACS may act as a secondary inducer in a positive feedback mechanism.
Although cytokinin and ENOD40 both negatively affect elongation growth, providing the possibility that their signalling pathways share common downstream components, no indication was found that interaction between these factors indeed occurs. This is based on the observations that ENOD40-provoked growth suppression does not require exogenous cytokinin, nor is cytokinin dose-dependent growth suppression significantly different in the transgenic and wild-type lines (Fig. 4C).
A major question now, is whether the observations made in this cellular model system can be extrapolated to the whole-plant level. Ectopic expression of GmENOD40 in Arabidopsis did not lead to severe changes in overall plant architecture, but led to a decrease in cell size of epidermal internode and leaf mesophyll cells (Guzzo et al., 2005). In addition, a subpopulation of protoplasts isolated from Arabidopsis cell suspension culture displayed reduced expansion growth after either transient expression of GmENOD40 or direct administration of GmENOD40 peptides (Guzzo et al., 2005). Thus, these data suggest that the phenotypic effect of ectopic expression of ENOD40 may depend on the cell type and/or environmental or developmental context. Upon inoculation of leguminous plants with rhizobia, ENOD40 is highly induced in cortical cells, several hours prior to the first cell division leading to nodule primordium formation, suggesting that ENOD40 is involved in the control over cortical cell division. In Medicago plants ectopically expressing MtENOD40, overexpression of MtENOD40 leads to proliferation of cells in the upper region of the root (Charon et al., 1997). However, upon inoculation of plants with the symbiont Sinorhizobium meliloti, cell proliferation is induced in the region close to the root tip (Charon et al., 1999), showing that overexpression of ENOD40 does not lead to cell division per se and that ENOD40 expression alone is not sufficient for nodule primordium formation. Likewise, in BY-2 cells overexpression of ENOD40 did not lead to an increase in cell division frequency. Thus, the phenotypic response to ectopic expression of ENOD40 may be dependent on the cellular context and/or on local action of other plant factors, such as phytohormones.
Since ENOD40 is highly induced and acts as a positive regulator during nodulation, the observation that ENOD40 overexpression leads to an acceleration of ethylene biosynthesis in BY-2 cells appears counter-intuitive to the function of ethylene as a negative regulator of nodulation during legume–Rhizobium interaction. It has been demonstrated that ethylene acts at a multitude of steps in the nodulation pathway, including nodule formation (Peters and Crist-Estes, 1989; Lee and LaRue, 1992); infection thread formation (Penmetsa and Cook, 1997); root hair deformation, early gene expression, and calcium spiking in response to Nod factor (Oldroyd et al., 2001), and regulation of the maintenance of nodule meristems in Sesbania rostrata (Fernandez-López et al., 1998). In addition, ethylene has a possible role in defining the position at which nodule primordia are initiated (Heidstra et al., 1997). Together, these observations have led to the hypothesis (Oldroyd et al., 2001) that ethylene could function as a dynamic regulator in the nodulation process by acting at an early point in Nod factor signalling at, or upstream of, calcium spiking in response to Nod factor, resulting in multiple ethylene-regulated developmental effects downstream in the Nod factor-dependent pathway. Alternatively, ethylene could inhibit several components of the nodulation pathway directly and independently (Oldroyd et al., 2001). Although the underlying mechanism remains unclear, the present studies in BY-2 cells suggest appropriate timing of ethylene biosynthesis as a critical factor for cellular responses, and indicate that ENOD40 may participate in the control over this timing. In the absence of studies describing the effect of either endogenous or ectopic expression of ENOD40 on ethylene biosynthesis, the existence of these interactions in whole-plant systems, or indeed during nodule development, still needs to be confirmed. Due to the diversity of possible responses induced by alterations of ethylene levels in whole-plant systems, the phenotypic outcome of such interactions is difficult to predict, and may be dependent on the cellular context. Nevertheless, it is conceivable that local expression of ENOD40 during early steps of the legume–Rhizobium interaction (Kouchi and Hata, 1993; Yang et al., 1993; Compaan et al., 2001) would provide the plant with a means to attenuate, in a dynamic manner, ethylene production, in keeping with the proposed role of ethylene as a dynamic regulator of cellular responses (Oldroyd et al., 2001) during nodulation. Therefore, effects of ENOD40 on the ethylene biosynthesis pathway could be considered as an important component of the complex regulatory pathway controlling nodule development.
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Acknowledgements |
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We thank Danny Geelen for kindly supplying the BY-2 cell suspension. We gratefully acknowledge Gerard van der Krogt for his valuable contribution to the BY-2 transformations, Véronique Storme for support with data analysis, and the excellent technical assistance of Ciska Braam and Maelle Lorvellec. We are thankful to Mark Hink and Jan-Willem Borst (Wageningen University Microscopy Center) for use of the microscope facilities. This work was supported by the Netherlands Organization for Scientific Research (NWO 805.49.004).
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Footnotes |
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* Present address: Department of Plant Systems Biology, VIB/Gent University, Technologiepark 927, 9052, Gent, Belgium.
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References |
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