Plant Cell Rep (2014) 33:1737–1744 DOI 10.1007/s00299-014-1652-1
ORIGINAL PAPER
Differential regulation of cytokinin oxidase genes and cytokinininduced auxin biosynthesis by cellular cytokinin level in transgenic poplars Young Im Choi • Eun Woon Noh • Hae Jung Kim Woong June Park
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Received: 8 April 2014 / Revised: 24 June 2014 / Accepted: 29 June 2014 / Published online: 22 July 2014 Ó Springer-Verlag Berlin Heidelberg 2014
Abstract Key message The present work with transgenic poplar lines producing varying levels of trans-zeatin suggests the existence of a switching threshold for triggering ckx gene expression or suppressing cytokinin-induced auxin. Abstract Cytokinins have an important role in growth and developmental processes of plants. Transgenic plants with varying levels of cellular cytokinin are convenient tools for studying its role in morphogenetic as well as molecular responses. In this work, the transgenic lines producing either high level of cellular trans-zeatin (HX lines) or moderate level (MX lines) were compared with regard to their cytokinin oxidase activities and cellular auxin content. The HX lines showed typical cytokinin phenotypes including leafy shoots and spontaneous shoot formation on hormone free medium. In contrast, the MX lines did not show any striking phenotypes. However, in leaf disk culture on hormone free medium, they regenerated roots and subsequently formed shoots from the roots. Determination of cellular IAA content revealed a significant increase in the level in MX lines but not in HX lines. Of nine cytokinin oxidase genes (ckx) examined by qPCR, five were activated in HX lines but not in MX lines. Among them, ckx4
Communicated by Prakash P. Kumar. Y. I. Choi (&) E. W. Noh H. J. Kim Forest Biotechnology Division, Korea Forest Research Institute, Suwon, Gyeonggi do 441-847, Republic of Korea e-mail: [email protected] W. J. Park Department of Molecular Biology, Institute of Nanosensor and Biotechnology, Dankook University, Youngin, Gyeonggi do 448-701, Republic of Korea
appeared to play a key role in maintaining cellular cytokinin level since it showed more than 1,000-fold increase in HX lines and in the leaf disks of untransformed control exposed to exogenous cytokinins. Although low level of cellular cytokinin did not induce the expression of ckx genes, it appeared to trigger cellular IAA biosynthesis. Keywords Tzs Cytokinin Cytokinin oxidase IAA biosynthesis Transgenic plants Poplar Abbreviations BAP 6-Benzylamino purine CKX Cytokinin oxidase IAA Indole-3-acetic acid qPCR Quantitative real-time PCR tzs Trans-zeatin secretion 2ip Isopentenyl adenine
Introduction Cytokinin together with auxin induces a range of morphogenetic responses in plants. A balance between them is most important for such responses (Skoog and Miller 1957). In addition, there appears to exist a threshold level for morphogenesis. In tissue culture experiments, it was demonstrated that while high levels of cytokinin led to both excessive shoot branching and poor root formation, the concentration below a certain level could not induce any morphogenetic responses (Komal 2011). In contrast, when the level was reduced beyond a certain level, it may trigger the onset of leaf senescence (Werner et al. 2003). Therefore,
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it is easily speculated that plants have some devices to maintain proper cellular cytokinin level. Recently, regulation dynamics of cellular cytokinin pool have been uncovered after feeding various cytokinins and then monitoring internal cytokinin metabolites (Lexa et al. 2003). Cytokinin oxidases (CKXs) are the mostwidely recognized enzymes regulating cellular cytokinin level in plants. It has been well documented that cellular CKX levels are associated with morphogenetic responses in plants. Using cytokinin deficient phenotypes, Werner et al. (2003) demonstrated that the major effects of cytokinin deficiency were reduction in shoot development, leading to dwarfed late-flowering plants, enhanced root growth, and altered reproductive development. Now it is well known that cellular cytokinin homeostasis is maintained both by new biosynthesis and by degradation (Jones et al. 2010; Werner et al. 2003). With the availability of transgenic technologies, cellular cytokinin level could be artificially manipulated by expression of biosynthetic genes (ipt or tzs) or metabolizing genes (ckx) (Motyka et al. 1996; Werner et al. 2003; Choi et al. 2009). Werner et al. (2003) demonstrated that transgenic Arabidopsis plants expressing each of six different members of the cytokinin oxidase/dehydrogenase (Atckx) gene family had increased cytokinin breakdown (30–45 % of wild-type cytokinin content). It was also shown that these cytokinin oxidases differing in catalytic properties seem to fine-tune cellular cytokinin pool (Schmu¨lling et al. 2003). Gu et al. (2010) summarized the tissue specific expression patterns of different ckx genes in rice, Arabidopsis and poplar. All these works suggest that CKXs play a key role in maintaining cytokinin homeostasis. However, it is still not clear the approximate threshold that triggers the induction of CKXs to reduce excessive cellular cytokinin level. Previously, we showed that our transgenic poplar lines produced varying levels of cellular zeatin when a bacterial tzs gene was expressed under 35S promoter of CaMV or AUX promoter of A. rhizogenes (Choi et al. 2009). A bacterial transzeatin secretion (tzs) gene driven by CaMV 35S promoter resulted in high transgene expression, a high level of cellular zeatin, and typical cytokinin induced phenotypes including multiple leafy shoots with thick stems and absence of roots in culture. In contrast, the tzs that was attached to AUX promoter did not induce much phenotype change in culture except forming roots from leaf disks on hormone-free medium, suggesting the involvement of interaction with auxin. Furthermore, the pAux-tzs transgenic lines produced about 20–30 % more biomass than did non-transgenic control in a single growing season in the nursery (Choi et al. 2009). Compared with moderately expressing pAUX-tzs lines (MX), high expressing p35S-tzs lines (HX) showed hundreds fold higher transcription of the transgene. However, their cellular cytokinin level was only several fold higher than that in MX lines.
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Plant Cell Rep (2014) 33:1737–1744 Table 1 The characteristics of the tzs transgenic poplar lines used in this study Line
Chimeric transgene
Cellular cytokinin content (nmol/g.f.w.) 0.11 ± 0.02e
Control MX-1
pAUX-tzs
0.34 ± 0.03de
MX-2 MX-3
pAUX-tzs pAUX-tzs
1.08 ± 0.03dc 0.39 ± 0.04de
HX-1
p35S-tzs
2.58 ± 0.08ab
HX-2
p35S-tzs
3.01 ± 0.18a
HX-3
p35S-tzs
1.86 ± 0.1bc
Means (±standard deviation) with the same letter are not significantly different at the 5 % p35S-tzs: tzs gene under 35S promoter of cauliflower mosaic virus (CaMV) pAUX-tzs: tzs gene under AUX promoter of Agrobacterium rhizogenes The transgenic lines and their trans-zeatin content were previously published (Choi et al. 2009)
Since trans-zeatin is speculated to be produced constitutively in the transgenic lines, there should exist some homeostatic mechanisms that maintain cellular cytokinin level within certain physiological limits. Therefore, it should be logical to test ckx expression to determine if they play any role in cytokinin over-producing transgenic lines. In poplar, nine ckx genes have now been known and thus allowed us to test their cellular expression level (Gu et al. 2010). Thus, the transgenic lines with different cellular zeatin levels may offer a good system to study the expression of each ckx gene in response to constitutively overproduced cellularcytokinin. In the present study, therefore, we examined the effects of the changes in cellular cytokinin level caused by the transgene expression on morphogenetic responses, cellular auxin level, and cytokinin oxidase activities to identify cause-effect relationships among them.
Materials and methods Plant materials Six cytokinin producing transgenic poplar lines (Populus alba 9 P. tremular var. glandulosa) were used in the present study. These include three HX lines in which a bacterial tzs gene was driven by CaMV 35S promoter and three MX lines with the same gene under AUX promoter of A. rhizogenes. The details of the production of transgenic poplars were described elsewhere (Choi et al. 2009) and their trans-zeatin content was summarized in Table 1. In short, the highly expressing (HX) phenotypes include short bush type leafy shoots even in the absence of exogenously
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provided cytokinin, lack of roots even in the supplement of auxin, spontaneous shoot regeneration from leaf disk culture on hormone-free medium. In contrast, the phenotypes of moderately expressing (MX) lines are a few normal looking axillary shoot formation and vigorous root growth. The transgenic lines were maintained by subculturing at 4 week intervals on hormone-free MS medium (Murashige and Skoog 1962) until use. Quantification of cellular auxin contents To determine the auxin level in the tzs transgenic lines, IAA was extracted and analyzed as described in Seo et al. (2009). The plantlets grown on hormone-free MS medium were harvested, weighed and extracted with 80 % ethanol. Deuteriumlabeled IAA (D5-IAA; Cambridge Isotopes) was added to the plant materials as an internal standard just before the extraction. The lipid components were allowed to precipitate for 16 h at 20 °C. The precipitates and debris were cleared out by centrifugation (17,6009g, 10 min, 4 °C). The extract was subjected to alkaline hydrolysis by boiling in 1 N NaOH to release the conjugated IAA for the measurement of total IAA. The IAA in the extract were pretreated with a C18 cartridge (Waters) and resolved on a reverse-phase HPLC column (Apollo C18, 5 mm; Alltech) set on a HPLC system (600E; Waters) equipped with a fluorescence detector (486; Waters). The IAA peaks were monitored with the emission at 360 nm (excitation at 286 nm) and the fractions were collected. The purified IAA was dried in a vacuum concentrator (Centrivap, Labconco, USA). The dried IAA was dissolved in a minimal volume of 10 % methanol and derivatized with (trimethylsilyl) diazomethane. The IAA was finally resolved on a capillary gas chromatography column (FactorFour VF-5 ms; Varian) set on a gas chromatography–mass spectrometry system (CP 3000, Saturn 2200; Varian). The fragmentation peak of 130 from IAA was compared with that of 135 from the internal standard and used for quantification. For both HX and MX lines, three independent transgenic lines with 3 replications were assayed.
Leaf disk culture To determine the effect of different cellular trans-zeatin levels on morphogenesis, ca. 5 mm 9 10 mm leaf disks were prepared from both HX and MX lines grown on hormone-free MS medium. The disks were then cultured on hormone-free MS medium for 4 weeks. Morphogenetic response was examined after 4 weeks in culture. The expression of ckx genes by exogenous hormones was also determined using leaf disks of untransformed control plants by culturing ten disks per petri dish containing solid MS medium with either cytokinin or auxin for 48 h. When assayed, the disks in a petri dish were pooled together and considered as
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a replicate. Each treatment consisted of three dishes (30 leaf disks). The effects of three different cytokinins (trans-zeatin, 2-isopenteny adenine (2ip), and 6-benzylamino purine (BAP) at three different levels (1, 5 and 25 nmol/g.f.w) were compared by quantitative realtime PCR (qPCR) method. qPCR analyzes of transgene and ckx genes To quantify the expression of the transgene tzs driven by the two different promoters, qPCR was performed using the total RNA isolated from both HX and MX lines. Specific primers for the transgene tzs sequence were based on Beaty et al. (1986) as follows: tzs F: 50 -TCT AGA CGA AAA TGT TAC TCC ATC TCA TCT AC-30 and tzs R: 50 GAG CTC ACC GAA TTC GCG TCA GCG T-30 . The qPCR was performed using the total RNA isolated from both transgenic lines and untransformed control plants. Nine poplar ckx genes (1–9) were used to monitor the response to cytokinins of either endogenously produced or exogenously provided (Genebank accession ckx1: XM_002300706, ckx2: XM_002304737, ckx3: XM_ 002307645, ckx4: XM_002308264, ckx5: XM_002308894, ckx6: XM_002309432, ckx7: XM_002310068, ckx8: XM_ 002323238, ckx9: XM_002332387). The specific primers were designed to amplify nine ckx genes as follows: ckx 1F: 50 -ACC CTT ATA GCC AGA AAT TCG-30 , ckx 1R: 50 -TCG TTT CAT CAA AGC CTC CA-30 , ckx 2F: 50 -TCG GAG AAG CAA AAT AGC GA-30 , ckx 2R: 50 -CCT TCA ATG TAG TCG AAC GTG-30 , ckx 3F: 50 -GAG GCT TTG ATG AAG AAC CTG-30 , ckx 3R: 50 -GTT TCC CAA AAT GCC CTT GA-30 , ckx 4F: 50 -TGG GAC CAG ACA CAT TGT TGA-30 , ckx4R: TCA TGG AAA ACA CCC TTG TTG-30 , ckx 5F: 50 -CAA CAA CTG GAG ATC CTC CTT30 , ckx 5R: 50 -ACT CTG TCC AGG AAT TCC ACA-30 , ckx 6F: 50 -TGG GAA GAT GTG TTA AAA CGA-30 , ckx 6R: 50 -GCA CCA AAA AAC AAT TCA CTG-30 , ckx 7F: 50 -AAA GGG GAG CTT GTG ACC TG-30 , ckx 7R: 50 TGA ACC TTC CAA ATA ATT GGC-30 , ckx 8F: 50 -AAA CAT GGA ATC CCT TCA GG-30 , ckx 8R: 50 -TGT TGT TAA TTT GTG GCC CA-30 , ckx 9F: 50 -ACA GTT GCA GCT AGA GGC CA-30 , and ckx 9R: 50 -CAA AGT ACC GCC AAC AGT TAA-30 . As an internal standard, poplar b-actin gene (ACT9: GenBank Accession XM_ 002331844.1) was used since the variation of the gene expression between treatments and clones in poplar was reported as very low (Regier and Frey 2010). Primers for bactin (ACT9) gene consist of ACT-9F: 50 -ACC ATC TCT CAT CGG AAT GGA A-30 and ACT-9R: 50 -AGG GCA GTG ATT TCC TTG CTC A-30 . The total RNA was isolated using TRI Reagent (Molecular Research Center, Cincinnati, OH, USA). The cDNA template was synthesized using a commercial RT–reagent kit (Takara Inc. Otsu, Japan). The procedure of the cDNA
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Fig. 1 IAA contents (nmol per gram fresh weight) in two different transgenic poplar lines with different cellular trans-zeatin levels. Con untransformed control, MX moderately expressing (pAUX-tzs) lines, and HX highly expressing (p35S-tzs) lines. The data of trans-zeatin content are from Choi et al. (2009) Means (±standard deviation) with the same letter are not significantly different at the 5 % of significance
synthesis was done according to the supplier’s manual. The reaction mixture contained 50 lM of oligo(dT) primer and 100 lM of random 6-mers, 1 lg of total RNA, and Primer RT mix. cDNA synthesis was done at 37 °C for 20 min followed by 10 s incubation at 85 °C. For realtime PCR, 1 ll of cDNA was amplified using a commercial amplification kit (SYBR premix Ex TaqII, Takara). Throughout the experiments, the PCR mixture contained (29) SYBR Premix Ex Taq II, 0.4 ll each of forward and reverse primers using triplet reaction for every treatment. For PCR amplification, cDNA templates were denatured for 30 s at 95 °C and then subjected to 40 cycles of denaturation at 95 °C for 10 s, annealing at 55 °C for 10 s and extension at 72 °C for 30 s. Final extension was performed at 72 °C for 5 min. To normalize the variation between cDNAs, the expression level of b-actin of poplar was used as housekeeping control. Means and SEs (n = 3) are present on the figures.
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Fig. 2 Morphogenetic responses of leaf disks from trans-zeatin producing transgenic poplar lines on hormone free medium. Whereas multiple leafy shoots regenerate from the leaf disks of HX plants, both roots and normal looking leaves were regenerated from those of MX plants. The leaf disks from control plants did not show any morphogenetic responses. Note that one p35S-tzs line formed vigorously growing callus instead of forming leafy shoots. Con untransformed control, MX moderately expressing (pAUX-tzs) lines, and HX highly expressing (p35S-tzs) lines
Results
control, HX lines did not show any increase in cellular auxin content, suggesting that higher cellular cytokinin content did not induce auxin biosynthesis. Thus, cellular cytokinin of less than 1 nmol/g.f.w could induce auxin biosynthesis (Table 1). In other words, cellular cytokinin level higher than 1.6 nmol/g.f.w did not seem to induce cellular auxin biosynthesis. As shown in Fig. 2, the leaf disks of different transgenic lines responded differently when cultured on hormone-free MS medium. The two HX lines (HX-1 and 2) formed 4–5 adventitious shoots from the edge of the leaf disks in the absence of any exogenous hormones. In contrast, the remaining HX line (HX-3) with less cellular zeatin and higher IAA than the other two lines formed one or two adventitious shoots which soon became surrounded by vigorous growing callus. The shoots from HX lines never developed any roots. In the case of MX lines (MX-1, 2, and 3) with their moderate cellular zeatin level, roots were first formed and then shoots were spontaneously developed from the roots in the absence of any exogenous hormones. The leaf disks from untransformed control plants did not form any roots or shoots at all either on hormone free medium. The morphogenetic responses appeared to be well matched to their cytokinin to auxin ratio shown in Fig. 1.
Cellular auxin contents and morphogenetic responses of the transgenic lines
Real-time PCR analyzes of tzs transgene in the transgenic plants
Auxin contents of transgenic lines ranged from 0.5 to 6.3 (nmol/g.f.w) (Fig. 1). Whereas auxin levels in MX lines were significantly higher than that in untransformed
The MX lines showed approximately 1,000 to 3,000-fold as high mRNA level of tzs gene as that of ß-actin gene used as an internal control (Fig. 3). In contrast, the expression level
Statistical analysis Data were analyzed by analysis of variance (ANOVA) followed by Duncan’s multiple range test (P B 0.05) using the SAS statistical program SAS 6.12 (SAS Institute, Cary, NC).
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Fig. 3 Relative expression rate of tzs in MX and HX transgenic poplar lines. The transcript level of tzs was normalized to the expression of b-actin gene. Since tzs is of bacterial origin, nontransgenic control plant does not have the transcript. Con untransformed control, MX moderately expressing (pAUX-tzs) lines, and HX highly expressing (p35S-tzs) lines. Means (±standard deviation) with the same letter are not significantly different at the 5 % of significance
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Fig. 4 Relative expression rate of different poplar cytokinin oxidase (ckx) genes in leaf disks of non-transgenic control plants 48 h after exposure to two different levels (5 and 25 nmol) of exogenous transzeatin. The transcript levels of ckxs were normalized to the expression level of b-actin gene. Means (±standard deviation) with the same letter are not significantly different at the 5 % of significance
of tzs mRNA in HX lines were 40,000–100,000 times higher than that of b-actin gene. Thus, HX lines showed 20–50 times higher in transgene expression than MX lines. Since tzs is of bacterial origin, untransformed control plants did not express the gene. The level of trans-zeatin in transgenic lines shown in Table 1 appeared to be matched well with the expression level shown by qPCR (Fig. 3). However, the magnitude of the expression revealed by qPCR did not proportionally translate into cellular transzeatin level (Table 1). Realtime PCR analyzes of ckx genes in the presence of exogenous cytokinin Since only small increase in cellular cytokinin level was observed even with very high transcription of tzs transgene, the involvement of cytokinin oxidases in trans-zeatin degradation was examined. For this, the leaf disks of untransformed control plants were harvested from 48 h culture after feeding either 5 or 25 nmol trans-zeatin and assayed for the expression of nine poplar ckx genes by qPCR. Of the 9 ckx genes, six appeared to be up-regulated ([2-fold) by exogenous trans-zeatin (Fig. 4). These include ckx 1, 3, 4, 7, 8 and 9. Both ckx1 and 3 showed more than a tenfold increase in the expression level upon exposure to 25 nmol trans-zeatin. However, the overall highest (3,000-fold) increase in the expression level was obtained by ckx4. Three ckx genes (7, 8, and 9) were modestly expressed. The remaining three genes (2, 5 and 6)
Fig. 5 Relative expression rate of cytokinin oxidase 4 (ckx4) genes in leaf disks of non-transgenic control plants 48 h after exposure to three different levels (1, 5 and 25 nmol) of cytokinins. The transcript levels were normalized to expression level of b-actin. Means (±standard deviation) with the same letter are not significantly different at the 5 % of significance
were not expressed or even suppressed. In addition, some genes including ckx 3, 7 and 9 that responded to 25 nmol trans-zeatin did not respond to low level (5 nmol). Thus, ckx4 was selected to further test its expression response to other cytokinins including BAP and 2ip. Figure 5 shows the expression levels of ckx4 gene after feeding three different levels of each hormone to the leaf disks of untransformed control plant. The gene was highly expressed in response to other cytokinins too. Among three cytokinins tested, trans-zeatin appeared to be the most active in ckx4 induction, which was followed by BAP and 2ip.
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Fig. 6 Relative expression rate of nine poplar cytokinin oxidase genes (ckx1 * 9) in transgenic lines. Con untransformed control, MX moderately expressing (pAUX-tzs) lines, and HX highly expressing
(p35S-tzs) lines. Means (±standard deviation) with the same letter are not significantly different at the 5 % of significance
Realtime PCR analyzes of ckx expression in transgenic lines
also the predominantly expressed ckx gene in transgenic lines. However, it was not expressed in MX lines at all, suggesting that it needs the presence of cellular cytokinin higher than certain level. Thus, it appeared to be the most active cytokinin degrading gene in poplar showing more than 1,000-fold increase in HX lines as well as in the zeatin-fed leaf disks of untransformed plants. ckx7 is the second most active gene next to ckx4 in HX lines. However, its expression level was two orders of magnitude smaller in the leaf disks of untransformed control plants exposed to exogenously supplied zeatin. The gene ckx 8 was modestly expressed and showed similar pattern with each other. Overall, eight ckx genes did not show any increase in the expression level in MX lines when compared with control plants, suggesting that constitutive presence of cellular cytokinin at modest level does not induce these genes.
The expression of nine poplar ckx genes was compared among transgenic lines (Fig. 6). Five ckx genes including ckx1, 3, 4, 7, and 8 were expressed in HX lines. However, only one of them, ckx1, was expressed in MX lines. Whereas the ckx1 gene was expressed 40 to 70-fold as high in HX lines as in untransformed control, it increased a mere 3–12-fold in MX lines. Three genes, ckx 2, 5, and 6, did not show any increase in the expression level and two of them, ckx 5 and 6, even appeared to be suppressed in transgenic lines. The lack of response in these genes to enhanced level of cellular zeatin is consistent with the results of the leaf disk experiments with exogenously provided cytokinins shown in Fig. 4. As in the case of exogenous cytokinin fed leaf disks, ckx 4 was
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Discussion The morphogenetic responses of the tzs transgenic lines in leaf disk culture matched well with their cellular hormone contents in that whereas the HX lines 1 and 2 containing high cellular zeatin and low IAA levels showed shoot regeneration from leaf disks, the HX line 3 with moderate levels of both zeatin and IAA produced vigorous growing callus from the disks (Figs. 1, 2). In contrast, the MX lines with their moderate cytokinin level, were found to contain surprisingly higher level of IAA than did HX lines or untransformed control. The higher IAA content in MX lines is consistent with root regenerating response from leaf disks of MX lines on hormone-free medium. Although the interaction between cytokinin and auxin is believed to play an important role in plant growth and development, it is still not well understood how their levels are coordinated in the process. There has been some evidence showing cytokinin stimulates auxin biosynthesis. Jones et al. (2010) demonstrated that cytokinin triggered auxin biosynthesis and proposed that a homeostatic feedback regulatory loop involving both auxin and cytokinin signaling act to maintain appropriate auxin and cytokinin concentrations in developing root and shoot tissues. Sun et al. (2003) also showed that increasing cytokinin levels via the estradiol induction of IPT8 in the transgenic line caused a rapid increase in the rate of indole-3-acetic acid (IAA) biosynthesis and in the levels of IAA. However, studying with transgenic tobacco expressing either cytokinin (ipt) or IAA (iaaM and iaaH) genes, Eklo¨f et al. (2000) demonstrated that over production of cytokinin lowered the cellular IAA level and vice versa. Beinsberger et al. (1991) also showed that ipt transgenic tobacco plants contained an increased endogenous cytokinin level in comparison with controls although no detectable changes in endogenous IAA level. In our transgenic lines, whereas HX lines with high cellular zeatin level did not show any change in cellular IAA level, MX lines with modest level of cytokinin contained high level of cellular IAA. Our results are consistent with those seemingly contradictory reports showing either effective or ineffective triggering of auxin biosynthesis by cytokinin (Jones et al. 2010; Eklo¨f et al. 2000; Beinsberger et al. 1991). Thus cytokinin induced auxin biosynthesis might occur in plants only when cytokinin at low level is present, suggesting a fine tuning mechanism to control auxin biosynthesis by cytokinin. In our data, a significant gap exists between the expression level revealed by qPCR and that of cellular zeatin in the transgenic lines. Despite big changes in transcription level, cellular zeatin levels showed minimum increase. This finding was consistent with previous work of Eklo¨f et al. (2000) showing that transgenic tobacco plants
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carrying both iaa and ipt genes contained IAA levels that were very similar to wild-type levels, although expression of the bacterial IAA biosynthesis genes was not reduced. It is now well established that cytokinin homeostasis is regulated by the rate of de novo synthesis and the rate of export and catabolism (Mok and Mok 2001). In our study, significant increase in ckx genes was observed in both transgenic lines and the leaf disks fed by exogenous cytokinin, indicating that CKXs play a central role in maintaining cellular cytokinin level in poplar. Of nine ckx genes in poplar, five appeared to be well expressed in both transgenic lines and leaf disks fed with exogenous cytokinins including zeatin, BAP and 2ip. Among them, ckx4 appeared to be the major ckx in poplar responsible for degradation of unusually high level cellular trans-zeatin. Except ckx1, most ckx genes did not show any increase in their expression in MX lines, suggesting that modest increase in cellular cytokinin level did not induce the genes. Thus, there seem to exist some thresholds for triggering ckx gene expression in poplar. In the case, 3 nmol trans-zeatin did not seem to be enough to trigger the expression of those genes. In conclusion, we used two different types of transgenic lines that differ in morphological responses in tissue culture and in their cellular trans-zeatin content and assessed the cellular auxin level as well as the expression level of both trans gene tzs and endogenous ckx genes in the plants. The two types of transgenic lines differ in their cellular auxin level since only moderately expressing lines showed increased cellular IAA content. Although the two types of transgenic lines differ less than fivefold from each other in their cellular zeatin level, they differ more than 100-fold in the transgene expression, indicating constitutive degradation of cellular zeatin by CKX. Among nine ckx genes tested, ckx4 appears to be the key gene responsible for degrading excessive cellular cytokinin that is constitutively produced in the HX lines. Acknowledgments YC, EN and HK were supported by Korea Forest Service and the research of WJP was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of education (NRF-20100023096). The authors declare no competing financial interests. Conflict of interest The authors whose names are listed immediately below report the following details of affiliation or involvement in an organization with a financial or non-financial interest in the subject matter or materials discussed in this manuscript.
References Beaty JS, Powell GK, Lica L, Regier DA, MacDonald EMS, Hommes NG, Morris RO (1986) Tzs, a nopaline Ti plasmid gene from Agrobacterium tumefaciens associated with trans-zeatin biosynthesis. Mol Gen Genet 203:274–280
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1744 Beinsberger SEI, Valcke RLM, Deblaere RY, Clijsters HMM, De Greef JA, Van Onckelen HA (1991) Effects of the introduction of Agrobacterium tumefaciens T-DNA ipt gene in Nicotiana tabacum L. cv. Petit Havana SR1 plant cells. Plant Cell Physiol 32:489–496 Choi YI, Noh EW, Choi KS (2009) Low level expression of prokaryotic tzs gene enhances growth performance of transgenic poplars. Trees 23:741–750. doi:10.1007/s00468-009-0316-1 ˚ stot C, Sitbon F, Moritz T, Olsson O, Sandberg G (2000) Eklo¨f S, A Transgenic tobacco plants co-expressing Agrobacterium iaa and ipt genes have wild-type hormone levels but display both auxinand cytokinin-overexpressing phenotypes. Plant J 23:279–284 Gu R, Fu J, Guo S, Duan F, Wang Z, Mi G, Yuan L (2010) Comparative expression and phylogenetic analysis of maize cytokinin dehydrogenase/oxidase (CKX) gene family. J Plant Growth Regul 29:428–440 Jones B, Gunnera SA, Petersson SV, Tarkowski P, Graham N, May S, Dolezal K, Sandberg G, Ljung K (2010) Cytokinin regulation of auxin synthesis in Arabidopsis involves a homeostatic feedback loop regulated via auxin and cytokinin signal transduction. Plant Cell 22:2956–2969 Komal R (2011) Effect of BAP and IAA on callus formation and plant regeneration in pointed gourd. Biotechnol Bioinf Bioeng 1:59–62 Lexa M, Genkov T, Malbeck J, Machakova I, Brzobohaty B (2003) Dynamics of endogenous cytokinin pools in tobacco seedlings: a modelling approach. Ann Bot 91:585–597 Mok DW, Mok MC (2001) Cytokinin metabolism and action. Annu Rev Plant Physiol Plant Mol Biol 52:89–118 Motyka V, Feiss M, Strand M, Kaminek M, Schmu¨lling T (1996) Changes in cytokinin content and cytokinin oxidase activity in
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Plant Cell Rep (2014) 33:1737–1744 response to derepression of ipt gene transcription in transgenic tobacco calli and plants. Plant Physiol 112:1035–1043 Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue culture. Physiol Plant 15:473–497 Regier N, Frey B (2010) Experimental comparison of relative RTqPCR quantification approaches for gene expression studies in poplar. BMC Mol Biol 11:57–58 Schmu¨lling T, Werner T, Riefler M, Krupkova´ E, Bartrina I, Manns Y (2003) Structure and function of cytokinin oxidase/dehydrogenase genes of maize, rice, Arabidopsis and other species. J Plant Res 116:241–252 Seo PJ, Xiang F, Qiao M, Park JY, Lee YN, Kim SG, Lee YH, Park WJ, Park CM (2009) The MYB96 transcription factor mediates abscisic acid signaling during drought stress response in Arabidopsis. Plant Physiol 151:275–289 Skoog F, Miller CO (1957) Chemical regulation of growth and organ formation in plant tissues cultured in vitro. Symp Soc Exp Biol 11:118–131 Sun J, Niu Q-W, Tarkowski P, Zheng B, Tarkowska D, Sandberg G, Chua N-H, Zuo J (2003) The Arabidopsis AtIPT8/PGA22 gene encodes an isopentenyl transferase that is involved in de novo cytokinin biosynthesis. Plant Physiol 131:167–176 Werner T, Motyka V, Laucou V, Smets R, Van Onckelen H, Schmu¨lling T (2003) Cytokinin-deficient transgenic Arabidopsis plants show multiple developmental alterations indicating opposite functions of cytokinins in the regulation of shoot and root meristem activity. Plant Cell 15:2532–2550
ORIGINAL PAPER
Differential regulation of cytokinin oxidase genes and cytokinininduced auxin biosynthesis by cellular cytokinin level in transgenic poplars Young Im Choi • Eun Woon Noh • Hae Jung Kim Woong June Park
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Received: 8 April 2014 / Revised: 24 June 2014 / Accepted: 29 June 2014 / Published online: 22 July 2014 Ó Springer-Verlag Berlin Heidelberg 2014
Abstract Key message The present work with transgenic poplar lines producing varying levels of trans-zeatin suggests the existence of a switching threshold for triggering ckx gene expression or suppressing cytokinin-induced auxin. Abstract Cytokinins have an important role in growth and developmental processes of plants. Transgenic plants with varying levels of cellular cytokinin are convenient tools for studying its role in morphogenetic as well as molecular responses. In this work, the transgenic lines producing either high level of cellular trans-zeatin (HX lines) or moderate level (MX lines) were compared with regard to their cytokinin oxidase activities and cellular auxin content. The HX lines showed typical cytokinin phenotypes including leafy shoots and spontaneous shoot formation on hormone free medium. In contrast, the MX lines did not show any striking phenotypes. However, in leaf disk culture on hormone free medium, they regenerated roots and subsequently formed shoots from the roots. Determination of cellular IAA content revealed a significant increase in the level in MX lines but not in HX lines. Of nine cytokinin oxidase genes (ckx) examined by qPCR, five were activated in HX lines but not in MX lines. Among them, ckx4
Communicated by Prakash P. Kumar. Y. I. Choi (&) E. W. Noh H. J. Kim Forest Biotechnology Division, Korea Forest Research Institute, Suwon, Gyeonggi do 441-847, Republic of Korea e-mail: [email protected] W. J. Park Department of Molecular Biology, Institute of Nanosensor and Biotechnology, Dankook University, Youngin, Gyeonggi do 448-701, Republic of Korea
appeared to play a key role in maintaining cellular cytokinin level since it showed more than 1,000-fold increase in HX lines and in the leaf disks of untransformed control exposed to exogenous cytokinins. Although low level of cellular cytokinin did not induce the expression of ckx genes, it appeared to trigger cellular IAA biosynthesis. Keywords Tzs Cytokinin Cytokinin oxidase IAA biosynthesis Transgenic plants Poplar Abbreviations BAP 6-Benzylamino purine CKX Cytokinin oxidase IAA Indole-3-acetic acid qPCR Quantitative real-time PCR tzs Trans-zeatin secretion 2ip Isopentenyl adenine
Introduction Cytokinin together with auxin induces a range of morphogenetic responses in plants. A balance between them is most important for such responses (Skoog and Miller 1957). In addition, there appears to exist a threshold level for morphogenesis. In tissue culture experiments, it was demonstrated that while high levels of cytokinin led to both excessive shoot branching and poor root formation, the concentration below a certain level could not induce any morphogenetic responses (Komal 2011). In contrast, when the level was reduced beyond a certain level, it may trigger the onset of leaf senescence (Werner et al. 2003). Therefore,
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it is easily speculated that plants have some devices to maintain proper cellular cytokinin level. Recently, regulation dynamics of cellular cytokinin pool have been uncovered after feeding various cytokinins and then monitoring internal cytokinin metabolites (Lexa et al. 2003). Cytokinin oxidases (CKXs) are the mostwidely recognized enzymes regulating cellular cytokinin level in plants. It has been well documented that cellular CKX levels are associated with morphogenetic responses in plants. Using cytokinin deficient phenotypes, Werner et al. (2003) demonstrated that the major effects of cytokinin deficiency were reduction in shoot development, leading to dwarfed late-flowering plants, enhanced root growth, and altered reproductive development. Now it is well known that cellular cytokinin homeostasis is maintained both by new biosynthesis and by degradation (Jones et al. 2010; Werner et al. 2003). With the availability of transgenic technologies, cellular cytokinin level could be artificially manipulated by expression of biosynthetic genes (ipt or tzs) or metabolizing genes (ckx) (Motyka et al. 1996; Werner et al. 2003; Choi et al. 2009). Werner et al. (2003) demonstrated that transgenic Arabidopsis plants expressing each of six different members of the cytokinin oxidase/dehydrogenase (Atckx) gene family had increased cytokinin breakdown (30–45 % of wild-type cytokinin content). It was also shown that these cytokinin oxidases differing in catalytic properties seem to fine-tune cellular cytokinin pool (Schmu¨lling et al. 2003). Gu et al. (2010) summarized the tissue specific expression patterns of different ckx genes in rice, Arabidopsis and poplar. All these works suggest that CKXs play a key role in maintaining cytokinin homeostasis. However, it is still not clear the approximate threshold that triggers the induction of CKXs to reduce excessive cellular cytokinin level. Previously, we showed that our transgenic poplar lines produced varying levels of cellular zeatin when a bacterial tzs gene was expressed under 35S promoter of CaMV or AUX promoter of A. rhizogenes (Choi et al. 2009). A bacterial transzeatin secretion (tzs) gene driven by CaMV 35S promoter resulted in high transgene expression, a high level of cellular zeatin, and typical cytokinin induced phenotypes including multiple leafy shoots with thick stems and absence of roots in culture. In contrast, the tzs that was attached to AUX promoter did not induce much phenotype change in culture except forming roots from leaf disks on hormone-free medium, suggesting the involvement of interaction with auxin. Furthermore, the pAux-tzs transgenic lines produced about 20–30 % more biomass than did non-transgenic control in a single growing season in the nursery (Choi et al. 2009). Compared with moderately expressing pAUX-tzs lines (MX), high expressing p35S-tzs lines (HX) showed hundreds fold higher transcription of the transgene. However, their cellular cytokinin level was only several fold higher than that in MX lines.
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Plant Cell Rep (2014) 33:1737–1744 Table 1 The characteristics of the tzs transgenic poplar lines used in this study Line
Chimeric transgene
Cellular cytokinin content (nmol/g.f.w.) 0.11 ± 0.02e
Control MX-1
pAUX-tzs
0.34 ± 0.03de
MX-2 MX-3
pAUX-tzs pAUX-tzs
1.08 ± 0.03dc 0.39 ± 0.04de
HX-1
p35S-tzs
2.58 ± 0.08ab
HX-2
p35S-tzs
3.01 ± 0.18a
HX-3
p35S-tzs
1.86 ± 0.1bc
Means (±standard deviation) with the same letter are not significantly different at the 5 % p35S-tzs: tzs gene under 35S promoter of cauliflower mosaic virus (CaMV) pAUX-tzs: tzs gene under AUX promoter of Agrobacterium rhizogenes The transgenic lines and their trans-zeatin content were previously published (Choi et al. 2009)
Since trans-zeatin is speculated to be produced constitutively in the transgenic lines, there should exist some homeostatic mechanisms that maintain cellular cytokinin level within certain physiological limits. Therefore, it should be logical to test ckx expression to determine if they play any role in cytokinin over-producing transgenic lines. In poplar, nine ckx genes have now been known and thus allowed us to test their cellular expression level (Gu et al. 2010). Thus, the transgenic lines with different cellular zeatin levels may offer a good system to study the expression of each ckx gene in response to constitutively overproduced cellularcytokinin. In the present study, therefore, we examined the effects of the changes in cellular cytokinin level caused by the transgene expression on morphogenetic responses, cellular auxin level, and cytokinin oxidase activities to identify cause-effect relationships among them.
Materials and methods Plant materials Six cytokinin producing transgenic poplar lines (Populus alba 9 P. tremular var. glandulosa) were used in the present study. These include three HX lines in which a bacterial tzs gene was driven by CaMV 35S promoter and three MX lines with the same gene under AUX promoter of A. rhizogenes. The details of the production of transgenic poplars were described elsewhere (Choi et al. 2009) and their trans-zeatin content was summarized in Table 1. In short, the highly expressing (HX) phenotypes include short bush type leafy shoots even in the absence of exogenously
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provided cytokinin, lack of roots even in the supplement of auxin, spontaneous shoot regeneration from leaf disk culture on hormone-free medium. In contrast, the phenotypes of moderately expressing (MX) lines are a few normal looking axillary shoot formation and vigorous root growth. The transgenic lines were maintained by subculturing at 4 week intervals on hormone-free MS medium (Murashige and Skoog 1962) until use. Quantification of cellular auxin contents To determine the auxin level in the tzs transgenic lines, IAA was extracted and analyzed as described in Seo et al. (2009). The plantlets grown on hormone-free MS medium were harvested, weighed and extracted with 80 % ethanol. Deuteriumlabeled IAA (D5-IAA; Cambridge Isotopes) was added to the plant materials as an internal standard just before the extraction. The lipid components were allowed to precipitate for 16 h at 20 °C. The precipitates and debris were cleared out by centrifugation (17,6009g, 10 min, 4 °C). The extract was subjected to alkaline hydrolysis by boiling in 1 N NaOH to release the conjugated IAA for the measurement of total IAA. The IAA in the extract were pretreated with a C18 cartridge (Waters) and resolved on a reverse-phase HPLC column (Apollo C18, 5 mm; Alltech) set on a HPLC system (600E; Waters) equipped with a fluorescence detector (486; Waters). The IAA peaks were monitored with the emission at 360 nm (excitation at 286 nm) and the fractions were collected. The purified IAA was dried in a vacuum concentrator (Centrivap, Labconco, USA). The dried IAA was dissolved in a minimal volume of 10 % methanol and derivatized with (trimethylsilyl) diazomethane. The IAA was finally resolved on a capillary gas chromatography column (FactorFour VF-5 ms; Varian) set on a gas chromatography–mass spectrometry system (CP 3000, Saturn 2200; Varian). The fragmentation peak of 130 from IAA was compared with that of 135 from the internal standard and used for quantification. For both HX and MX lines, three independent transgenic lines with 3 replications were assayed.
Leaf disk culture To determine the effect of different cellular trans-zeatin levels on morphogenesis, ca. 5 mm 9 10 mm leaf disks were prepared from both HX and MX lines grown on hormone-free MS medium. The disks were then cultured on hormone-free MS medium for 4 weeks. Morphogenetic response was examined after 4 weeks in culture. The expression of ckx genes by exogenous hormones was also determined using leaf disks of untransformed control plants by culturing ten disks per petri dish containing solid MS medium with either cytokinin or auxin for 48 h. When assayed, the disks in a petri dish were pooled together and considered as
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a replicate. Each treatment consisted of three dishes (30 leaf disks). The effects of three different cytokinins (trans-zeatin, 2-isopenteny adenine (2ip), and 6-benzylamino purine (BAP) at three different levels (1, 5 and 25 nmol/g.f.w) were compared by quantitative realtime PCR (qPCR) method. qPCR analyzes of transgene and ckx genes To quantify the expression of the transgene tzs driven by the two different promoters, qPCR was performed using the total RNA isolated from both HX and MX lines. Specific primers for the transgene tzs sequence were based on Beaty et al. (1986) as follows: tzs F: 50 -TCT AGA CGA AAA TGT TAC TCC ATC TCA TCT AC-30 and tzs R: 50 GAG CTC ACC GAA TTC GCG TCA GCG T-30 . The qPCR was performed using the total RNA isolated from both transgenic lines and untransformed control plants. Nine poplar ckx genes (1–9) were used to monitor the response to cytokinins of either endogenously produced or exogenously provided (Genebank accession ckx1: XM_002300706, ckx2: XM_002304737, ckx3: XM_ 002307645, ckx4: XM_002308264, ckx5: XM_002308894, ckx6: XM_002309432, ckx7: XM_002310068, ckx8: XM_ 002323238, ckx9: XM_002332387). The specific primers were designed to amplify nine ckx genes as follows: ckx 1F: 50 -ACC CTT ATA GCC AGA AAT TCG-30 , ckx 1R: 50 -TCG TTT CAT CAA AGC CTC CA-30 , ckx 2F: 50 -TCG GAG AAG CAA AAT AGC GA-30 , ckx 2R: 50 -CCT TCA ATG TAG TCG AAC GTG-30 , ckx 3F: 50 -GAG GCT TTG ATG AAG AAC CTG-30 , ckx 3R: 50 -GTT TCC CAA AAT GCC CTT GA-30 , ckx 4F: 50 -TGG GAC CAG ACA CAT TGT TGA-30 , ckx4R: TCA TGG AAA ACA CCC TTG TTG-30 , ckx 5F: 50 -CAA CAA CTG GAG ATC CTC CTT30 , ckx 5R: 50 -ACT CTG TCC AGG AAT TCC ACA-30 , ckx 6F: 50 -TGG GAA GAT GTG TTA AAA CGA-30 , ckx 6R: 50 -GCA CCA AAA AAC AAT TCA CTG-30 , ckx 7F: 50 -AAA GGG GAG CTT GTG ACC TG-30 , ckx 7R: 50 TGA ACC TTC CAA ATA ATT GGC-30 , ckx 8F: 50 -AAA CAT GGA ATC CCT TCA GG-30 , ckx 8R: 50 -TGT TGT TAA TTT GTG GCC CA-30 , ckx 9F: 50 -ACA GTT GCA GCT AGA GGC CA-30 , and ckx 9R: 50 -CAA AGT ACC GCC AAC AGT TAA-30 . As an internal standard, poplar b-actin gene (ACT9: GenBank Accession XM_ 002331844.1) was used since the variation of the gene expression between treatments and clones in poplar was reported as very low (Regier and Frey 2010). Primers for bactin (ACT9) gene consist of ACT-9F: 50 -ACC ATC TCT CAT CGG AAT GGA A-30 and ACT-9R: 50 -AGG GCA GTG ATT TCC TTG CTC A-30 . The total RNA was isolated using TRI Reagent (Molecular Research Center, Cincinnati, OH, USA). The cDNA template was synthesized using a commercial RT–reagent kit (Takara Inc. Otsu, Japan). The procedure of the cDNA
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Fig. 1 IAA contents (nmol per gram fresh weight) in two different transgenic poplar lines with different cellular trans-zeatin levels. Con untransformed control, MX moderately expressing (pAUX-tzs) lines, and HX highly expressing (p35S-tzs) lines. The data of trans-zeatin content are from Choi et al. (2009) Means (±standard deviation) with the same letter are not significantly different at the 5 % of significance
synthesis was done according to the supplier’s manual. The reaction mixture contained 50 lM of oligo(dT) primer and 100 lM of random 6-mers, 1 lg of total RNA, and Primer RT mix. cDNA synthesis was done at 37 °C for 20 min followed by 10 s incubation at 85 °C. For realtime PCR, 1 ll of cDNA was amplified using a commercial amplification kit (SYBR premix Ex TaqII, Takara). Throughout the experiments, the PCR mixture contained (29) SYBR Premix Ex Taq II, 0.4 ll each of forward and reverse primers using triplet reaction for every treatment. For PCR amplification, cDNA templates were denatured for 30 s at 95 °C and then subjected to 40 cycles of denaturation at 95 °C for 10 s, annealing at 55 °C for 10 s and extension at 72 °C for 30 s. Final extension was performed at 72 °C for 5 min. To normalize the variation between cDNAs, the expression level of b-actin of poplar was used as housekeeping control. Means and SEs (n = 3) are present on the figures.
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Fig. 2 Morphogenetic responses of leaf disks from trans-zeatin producing transgenic poplar lines on hormone free medium. Whereas multiple leafy shoots regenerate from the leaf disks of HX plants, both roots and normal looking leaves were regenerated from those of MX plants. The leaf disks from control plants did not show any morphogenetic responses. Note that one p35S-tzs line formed vigorously growing callus instead of forming leafy shoots. Con untransformed control, MX moderately expressing (pAUX-tzs) lines, and HX highly expressing (p35S-tzs) lines
Results
control, HX lines did not show any increase in cellular auxin content, suggesting that higher cellular cytokinin content did not induce auxin biosynthesis. Thus, cellular cytokinin of less than 1 nmol/g.f.w could induce auxin biosynthesis (Table 1). In other words, cellular cytokinin level higher than 1.6 nmol/g.f.w did not seem to induce cellular auxin biosynthesis. As shown in Fig. 2, the leaf disks of different transgenic lines responded differently when cultured on hormone-free MS medium. The two HX lines (HX-1 and 2) formed 4–5 adventitious shoots from the edge of the leaf disks in the absence of any exogenous hormones. In contrast, the remaining HX line (HX-3) with less cellular zeatin and higher IAA than the other two lines formed one or two adventitious shoots which soon became surrounded by vigorous growing callus. The shoots from HX lines never developed any roots. In the case of MX lines (MX-1, 2, and 3) with their moderate cellular zeatin level, roots were first formed and then shoots were spontaneously developed from the roots in the absence of any exogenous hormones. The leaf disks from untransformed control plants did not form any roots or shoots at all either on hormone free medium. The morphogenetic responses appeared to be well matched to their cytokinin to auxin ratio shown in Fig. 1.
Cellular auxin contents and morphogenetic responses of the transgenic lines
Real-time PCR analyzes of tzs transgene in the transgenic plants
Auxin contents of transgenic lines ranged from 0.5 to 6.3 (nmol/g.f.w) (Fig. 1). Whereas auxin levels in MX lines were significantly higher than that in untransformed
The MX lines showed approximately 1,000 to 3,000-fold as high mRNA level of tzs gene as that of ß-actin gene used as an internal control (Fig. 3). In contrast, the expression level
Statistical analysis Data were analyzed by analysis of variance (ANOVA) followed by Duncan’s multiple range test (P B 0.05) using the SAS statistical program SAS 6.12 (SAS Institute, Cary, NC).
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Fig. 3 Relative expression rate of tzs in MX and HX transgenic poplar lines. The transcript level of tzs was normalized to the expression of b-actin gene. Since tzs is of bacterial origin, nontransgenic control plant does not have the transcript. Con untransformed control, MX moderately expressing (pAUX-tzs) lines, and HX highly expressing (p35S-tzs) lines. Means (±standard deviation) with the same letter are not significantly different at the 5 % of significance
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Fig. 4 Relative expression rate of different poplar cytokinin oxidase (ckx) genes in leaf disks of non-transgenic control plants 48 h after exposure to two different levels (5 and 25 nmol) of exogenous transzeatin. The transcript levels of ckxs were normalized to the expression level of b-actin gene. Means (±standard deviation) with the same letter are not significantly different at the 5 % of significance
of tzs mRNA in HX lines were 40,000–100,000 times higher than that of b-actin gene. Thus, HX lines showed 20–50 times higher in transgene expression than MX lines. Since tzs is of bacterial origin, untransformed control plants did not express the gene. The level of trans-zeatin in transgenic lines shown in Table 1 appeared to be matched well with the expression level shown by qPCR (Fig. 3). However, the magnitude of the expression revealed by qPCR did not proportionally translate into cellular transzeatin level (Table 1). Realtime PCR analyzes of ckx genes in the presence of exogenous cytokinin Since only small increase in cellular cytokinin level was observed even with very high transcription of tzs transgene, the involvement of cytokinin oxidases in trans-zeatin degradation was examined. For this, the leaf disks of untransformed control plants were harvested from 48 h culture after feeding either 5 or 25 nmol trans-zeatin and assayed for the expression of nine poplar ckx genes by qPCR. Of the 9 ckx genes, six appeared to be up-regulated ([2-fold) by exogenous trans-zeatin (Fig. 4). These include ckx 1, 3, 4, 7, 8 and 9. Both ckx1 and 3 showed more than a tenfold increase in the expression level upon exposure to 25 nmol trans-zeatin. However, the overall highest (3,000-fold) increase in the expression level was obtained by ckx4. Three ckx genes (7, 8, and 9) were modestly expressed. The remaining three genes (2, 5 and 6)
Fig. 5 Relative expression rate of cytokinin oxidase 4 (ckx4) genes in leaf disks of non-transgenic control plants 48 h after exposure to three different levels (1, 5 and 25 nmol) of cytokinins. The transcript levels were normalized to expression level of b-actin. Means (±standard deviation) with the same letter are not significantly different at the 5 % of significance
were not expressed or even suppressed. In addition, some genes including ckx 3, 7 and 9 that responded to 25 nmol trans-zeatin did not respond to low level (5 nmol). Thus, ckx4 was selected to further test its expression response to other cytokinins including BAP and 2ip. Figure 5 shows the expression levels of ckx4 gene after feeding three different levels of each hormone to the leaf disks of untransformed control plant. The gene was highly expressed in response to other cytokinins too. Among three cytokinins tested, trans-zeatin appeared to be the most active in ckx4 induction, which was followed by BAP and 2ip.
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Fig. 6 Relative expression rate of nine poplar cytokinin oxidase genes (ckx1 * 9) in transgenic lines. Con untransformed control, MX moderately expressing (pAUX-tzs) lines, and HX highly expressing
(p35S-tzs) lines. Means (±standard deviation) with the same letter are not significantly different at the 5 % of significance
Realtime PCR analyzes of ckx expression in transgenic lines
also the predominantly expressed ckx gene in transgenic lines. However, it was not expressed in MX lines at all, suggesting that it needs the presence of cellular cytokinin higher than certain level. Thus, it appeared to be the most active cytokinin degrading gene in poplar showing more than 1,000-fold increase in HX lines as well as in the zeatin-fed leaf disks of untransformed plants. ckx7 is the second most active gene next to ckx4 in HX lines. However, its expression level was two orders of magnitude smaller in the leaf disks of untransformed control plants exposed to exogenously supplied zeatin. The gene ckx 8 was modestly expressed and showed similar pattern with each other. Overall, eight ckx genes did not show any increase in the expression level in MX lines when compared with control plants, suggesting that constitutive presence of cellular cytokinin at modest level does not induce these genes.
The expression of nine poplar ckx genes was compared among transgenic lines (Fig. 6). Five ckx genes including ckx1, 3, 4, 7, and 8 were expressed in HX lines. However, only one of them, ckx1, was expressed in MX lines. Whereas the ckx1 gene was expressed 40 to 70-fold as high in HX lines as in untransformed control, it increased a mere 3–12-fold in MX lines. Three genes, ckx 2, 5, and 6, did not show any increase in the expression level and two of them, ckx 5 and 6, even appeared to be suppressed in transgenic lines. The lack of response in these genes to enhanced level of cellular zeatin is consistent with the results of the leaf disk experiments with exogenously provided cytokinins shown in Fig. 4. As in the case of exogenous cytokinin fed leaf disks, ckx 4 was
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Discussion The morphogenetic responses of the tzs transgenic lines in leaf disk culture matched well with their cellular hormone contents in that whereas the HX lines 1 and 2 containing high cellular zeatin and low IAA levels showed shoot regeneration from leaf disks, the HX line 3 with moderate levels of both zeatin and IAA produced vigorous growing callus from the disks (Figs. 1, 2). In contrast, the MX lines with their moderate cytokinin level, were found to contain surprisingly higher level of IAA than did HX lines or untransformed control. The higher IAA content in MX lines is consistent with root regenerating response from leaf disks of MX lines on hormone-free medium. Although the interaction between cytokinin and auxin is believed to play an important role in plant growth and development, it is still not well understood how their levels are coordinated in the process. There has been some evidence showing cytokinin stimulates auxin biosynthesis. Jones et al. (2010) demonstrated that cytokinin triggered auxin biosynthesis and proposed that a homeostatic feedback regulatory loop involving both auxin and cytokinin signaling act to maintain appropriate auxin and cytokinin concentrations in developing root and shoot tissues. Sun et al. (2003) also showed that increasing cytokinin levels via the estradiol induction of IPT8 in the transgenic line caused a rapid increase in the rate of indole-3-acetic acid (IAA) biosynthesis and in the levels of IAA. However, studying with transgenic tobacco expressing either cytokinin (ipt) or IAA (iaaM and iaaH) genes, Eklo¨f et al. (2000) demonstrated that over production of cytokinin lowered the cellular IAA level and vice versa. Beinsberger et al. (1991) also showed that ipt transgenic tobacco plants contained an increased endogenous cytokinin level in comparison with controls although no detectable changes in endogenous IAA level. In our transgenic lines, whereas HX lines with high cellular zeatin level did not show any change in cellular IAA level, MX lines with modest level of cytokinin contained high level of cellular IAA. Our results are consistent with those seemingly contradictory reports showing either effective or ineffective triggering of auxin biosynthesis by cytokinin (Jones et al. 2010; Eklo¨f et al. 2000; Beinsberger et al. 1991). Thus cytokinin induced auxin biosynthesis might occur in plants only when cytokinin at low level is present, suggesting a fine tuning mechanism to control auxin biosynthesis by cytokinin. In our data, a significant gap exists between the expression level revealed by qPCR and that of cellular zeatin in the transgenic lines. Despite big changes in transcription level, cellular zeatin levels showed minimum increase. This finding was consistent with previous work of Eklo¨f et al. (2000) showing that transgenic tobacco plants
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carrying both iaa and ipt genes contained IAA levels that were very similar to wild-type levels, although expression of the bacterial IAA biosynthesis genes was not reduced. It is now well established that cytokinin homeostasis is regulated by the rate of de novo synthesis and the rate of export and catabolism (Mok and Mok 2001). In our study, significant increase in ckx genes was observed in both transgenic lines and the leaf disks fed by exogenous cytokinin, indicating that CKXs play a central role in maintaining cellular cytokinin level in poplar. Of nine ckx genes in poplar, five appeared to be well expressed in both transgenic lines and leaf disks fed with exogenous cytokinins including zeatin, BAP and 2ip. Among them, ckx4 appeared to be the major ckx in poplar responsible for degradation of unusually high level cellular trans-zeatin. Except ckx1, most ckx genes did not show any increase in their expression in MX lines, suggesting that modest increase in cellular cytokinin level did not induce the genes. Thus, there seem to exist some thresholds for triggering ckx gene expression in poplar. In the case, 3 nmol trans-zeatin did not seem to be enough to trigger the expression of those genes. In conclusion, we used two different types of transgenic lines that differ in morphological responses in tissue culture and in their cellular trans-zeatin content and assessed the cellular auxin level as well as the expression level of both trans gene tzs and endogenous ckx genes in the plants. The two types of transgenic lines differ in their cellular auxin level since only moderately expressing lines showed increased cellular IAA content. Although the two types of transgenic lines differ less than fivefold from each other in their cellular zeatin level, they differ more than 100-fold in the transgene expression, indicating constitutive degradation of cellular zeatin by CKX. Among nine ckx genes tested, ckx4 appears to be the key gene responsible for degrading excessive cellular cytokinin that is constitutively produced in the HX lines. Acknowledgments YC, EN and HK were supported by Korea Forest Service and the research of WJP was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of education (NRF-20100023096). The authors declare no competing financial interests. Conflict of interest The authors whose names are listed immediately below report the following details of affiliation or involvement in an organization with a financial or non-financial interest in the subject matter or materials discussed in this manuscript.
References Beaty JS, Powell GK, Lica L, Regier DA, MacDonald EMS, Hommes NG, Morris RO (1986) Tzs, a nopaline Ti plasmid gene from Agrobacterium tumefaciens associated with trans-zeatin biosynthesis. Mol Gen Genet 203:274–280
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1744 Beinsberger SEI, Valcke RLM, Deblaere RY, Clijsters HMM, De Greef JA, Van Onckelen HA (1991) Effects of the introduction of Agrobacterium tumefaciens T-DNA ipt gene in Nicotiana tabacum L. cv. Petit Havana SR1 plant cells. Plant Cell Physiol 32:489–496 Choi YI, Noh EW, Choi KS (2009) Low level expression of prokaryotic tzs gene enhances growth performance of transgenic poplars. Trees 23:741–750. doi:10.1007/s00468-009-0316-1 ˚ stot C, Sitbon F, Moritz T, Olsson O, Sandberg G (2000) Eklo¨f S, A Transgenic tobacco plants co-expressing Agrobacterium iaa and ipt genes have wild-type hormone levels but display both auxinand cytokinin-overexpressing phenotypes. Plant J 23:279–284 Gu R, Fu J, Guo S, Duan F, Wang Z, Mi G, Yuan L (2010) Comparative expression and phylogenetic analysis of maize cytokinin dehydrogenase/oxidase (CKX) gene family. J Plant Growth Regul 29:428–440 Jones B, Gunnera SA, Petersson SV, Tarkowski P, Graham N, May S, Dolezal K, Sandberg G, Ljung K (2010) Cytokinin regulation of auxin synthesis in Arabidopsis involves a homeostatic feedback loop regulated via auxin and cytokinin signal transduction. Plant Cell 22:2956–2969 Komal R (2011) Effect of BAP and IAA on callus formation and plant regeneration in pointed gourd. Biotechnol Bioinf Bioeng 1:59–62 Lexa M, Genkov T, Malbeck J, Machakova I, Brzobohaty B (2003) Dynamics of endogenous cytokinin pools in tobacco seedlings: a modelling approach. Ann Bot 91:585–597 Mok DW, Mok MC (2001) Cytokinin metabolism and action. Annu Rev Plant Physiol Plant Mol Biol 52:89–118 Motyka V, Feiss M, Strand M, Kaminek M, Schmu¨lling T (1996) Changes in cytokinin content and cytokinin oxidase activity in
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Plant Cell Rep (2014) 33:1737–1744 response to derepression of ipt gene transcription in transgenic tobacco calli and plants. Plant Physiol 112:1035–1043 Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue culture. Physiol Plant 15:473–497 Regier N, Frey B (2010) Experimental comparison of relative RTqPCR quantification approaches for gene expression studies in poplar. BMC Mol Biol 11:57–58 Schmu¨lling T, Werner T, Riefler M, Krupkova´ E, Bartrina I, Manns Y (2003) Structure and function of cytokinin oxidase/dehydrogenase genes of maize, rice, Arabidopsis and other species. J Plant Res 116:241–252 Seo PJ, Xiang F, Qiao M, Park JY, Lee YN, Kim SG, Lee YH, Park WJ, Park CM (2009) The MYB96 transcription factor mediates abscisic acid signaling during drought stress response in Arabidopsis. Plant Physiol 151:275–289 Skoog F, Miller CO (1957) Chemical regulation of growth and organ formation in plant tissues cultured in vitro. Symp Soc Exp Biol 11:118–131 Sun J, Niu Q-W, Tarkowski P, Zheng B, Tarkowska D, Sandberg G, Chua N-H, Zuo J (2003) The Arabidopsis AtIPT8/PGA22 gene encodes an isopentenyl transferase that is involved in de novo cytokinin biosynthesis. Plant Physiol 131:167–176 Werner T, Motyka V, Laucou V, Smets R, Van Onckelen H, Schmu¨lling T (2003) Cytokinin-deficient transgenic Arabidopsis plants show multiple developmental alterations indicating opposite functions of cytokinins in the regulation of shoot and root meristem activity. Plant Cell 15:2532–2550
