Journal of Experimental Botany, Vol. 53, No. 376, pp. 1899-1907,
September 1, 2002
© 2002 Oxford University Press
Isolation and characterization of the orchid cytokinin oxidase DSCKX1 promoter
Received 7 January 2002; Accepted 11 June 2002
Plant Growth and Development Laboratory, Department of Biological Sciences, Faculty of Science, National University of Singapore, 10 Kent Ridge Crescent, Singapore 119260, Republic of Singapore
Abbreviations: BA, benzylaminopurine; CaMV, cauliflower mosaic virus; GUS, ß-glucuronidase; iP, isopentenyl adenine; iPA, isopentenyl adenosine; MS, Murashige–Skoog; PLBs, protocorm-like-bodies; 5'-RACE, 5'-rapid amplification of cDNA ends; ZR, zeatin riboside.
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Abstract |
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The orchid DSCKX1 is a new member of the cytokinin oxidase gene family, which catalyses the degradation of cytokinins bearing unsaturated isoprenoid side chains. A 3.7 kb fragment upstream of the DSCKX1 coding region was isolated, sequenced and characterized by deletion analysis of DSCKX1::ß-glucuronidase gene fusions using transient orchid and stable Arabidopsis transformation systems. Functional analysis of 5' deletions defined the 5'-upstream region that directs the expression in distinct tissues. Regulatory elements affecting the cytokinin induction of the DSCKX1 gene have also been delineated
Key words: Key words: Cytokinin oxidase, orchid, regulation.
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Introduction |
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TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
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The degradative metabolism of cytokinins is an important process that controls the levels of cytokinin active forms and their distribution in plant tissues. It appears to be due, in large part, to the activity of a specific enzyme, cytokinin oxidase. Multiple forms of cytokinin oxidase have been identified and characterized in various plants, such as maize (Burch and Horgan, 1989), Phaseolus (Kamínek and Armstrong, 1990), wheat (Laloue and Fox, 1989), tobacco (Motyka and Kamínek, 1992), and Vinca rosea (McGaw and Horgan, 1983). Isolation of the cDNAs encoding cytokinin oxidase from maize and Arabidopsis has initiated the investigation of the cytokinin oxidase gene at the molecular level (Houba-Hérin et al., 1999; Morris et al., 1999; Bilyeu et al., 2001).
A novel cytokinin oxidase gene, DSCKX1 (accession no. AJ294542), has previously been isolated and characterized by mRNA differential display from shoot apices of Dendrobium Sonia cultured in the presence of benzylaminopurine (BA) (Yang et al., 2002). DSCKX1 is regulated by cytokinins, which is consistent with the physiological studies of cytokinin oxidase in other species (Chatfield and Armstrong, 1986; Kamínek and Armstrong, 1990; Motyka and Kamínek, 1990). This suggests that there are common mechanisms involving cytokinin oxidases in the regulation of cytokinin activities in different species. Therefore, the investigation of the regulatory mechanism of any of the cytokinin oxidase genes will help in understanding how its counterparts are controlled in the other plants. Interactions between trans-acting factors and cis-acting regulatory sequences in plant promoters need to be determined by the regulation of plant gene expression. So far, there is no report on the characterization of cis-acting elements and the related trans-acting factors involved in the regulation of cytokinin oxidase genes. Using transient expression in orchid and stable expression in Arabidopsis, the cis-acting elements of DSCKX1 have been investigated. Furthermore, possible regulatory regions affecting cytokinin induction of the DSCKX1 gene have also been delineated.
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Materials and methods |
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TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
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Plant materials and growth conditions
The protocorm-like-bodies (PLBs) of Dendrobium Sonia, a hybrid of Dendrobium Caesarx Dendrobium Tomie Drake, were cultured on liquid modified KC (Knudson, 1946) medium supplemented with 2% (w/v) sucrose, 15% (v/v) coconut water, 25 µM BA, and grown in a 16/8 h light/dark cycle with fluorescent light (35 µmol m–2 s–1) at 25 °C on rotary shakers at 120 rpm.
Plants of Arabidopsis thaliana (Columbia ecotype) were grown in a growth chamber at 22 °C under fluorescent white light (100 µmol m–2 s–1) in 16/8 h light/dark cycle, and 70% relative humidity. Arabidopsis seeds were grown on Petri dishes containing half-strength Murashige–Skoog (MS) medium (Murashige and Skoog, 1962; GIBCO BRL) supplemented with 2% sucrose and 0.3% Phytagel (Sigma), or potted in soil.
Isolation of DSCKX1 putative promoter by GenomeWalker DNA Walking
The 5'-flanking region of the DSCKX1 gene was isolated according to the instructions of Universal GenomeWalkerTM Kit (Clontech Laboratories, Palo Alto, CA) with some modifications. Genomic DNA was extracted from young orchid leaves by the method described previously (Yu and Goh, 2000). Isolated genomic DNA was digested with five restriction enzymes (DraI, EcoRV, PvuII, ScaI, and StuI, respectively) to create blunt-end fragments that were then ligated to a GenomeWalker adaptor to produce five respective GenomeWalker libraries.
The 3.73 kb DSCKX1 putative promoter fragment (–3724 to +4, accession no. AJ294543) was isolated by three successive PCR-based DNA walkings in GenomeWalker libraries. The primary PCR was performed with a gene-specific primer (GSP1; Table 1; Fig. 1B) and the outer adaptor primer (AP1) using the GeneAmp PCR System 2400 (Perkin-Elmer Applied Biosystems). The amplifications began with 5 cycles of 94 °C for 5 s and 72 °C for 3 min, followed by 30 cycles of 94 °C for 5 s and 68 °C for 3 min, and a final extension at 68 °C for 7 min. For each round of genome walking, the diluted primary PCR products served as templates for the secondary ‘nested’ PCR with a nested gene-specific primer (GSP2) and the nested adaptor primer (AP2). The secondary PCR products were analysed on agarose gels. The major bands were purified from gels with QIAEXII Gel Extraction Kit (Qiagen), cloned into pGEMT Easy Vector (Promega, Madison, WI) and sequenced. The primers used in the second and third rounds of walking were subsequently synthesized based on the sequences of secondary PCR products of previous rounds (Table 1).
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Identification of the transcription start site by 5'-rapid amplification of cDNA ends (5'-RACE)
5'-RACE was performed with total RNA extracted from the shoot apices of plantlets cultured in KC medium with 25 µM BA following the protocol supplied with the SMARTTM RACE cDNA Amplification Kit (Clontech Laboratories). 5'-RACE PCR was successively performed with two nested reverse DSCKX1 gene specific primers DSC1-GSP1 and DSC1-GSP2 (Table 1; Fig. 1B). The resulting amplified fragments were subsequently cloned and sequenced. Five positive clones were sequenced in order to map the transcription start site.
Sequencing and sequence analysis
Sequencing was carried out with the Big DyeTM Terminator Cycle Sequencing Ready Reaction kit and the model 377 automated sequencer (Perkin-Elmer Applied Biosystems, CA). Data about protein binding factors were obtained through the TRANSFAC (http://www.transfac.gbf.de/cgi-bin/matSearch/matsearch.pl) and PLACE (http://www.dna.affrc.go.jp/htdocs/PLACE/) databases.
Construction of chimeric reporter gene fusions
The DSCKX1 putative promoter deletion derivatives were transcriptionally fused with the ß-glucuronidase (GUS) reporter gene by cloning the various 5' deletion fragments into the polylinker region of the binary vector pBI101 (Clontech Laboratories) upstream of a promoterless GUS gene cassette.
The 3.73 kb and 2.2 kb putative promoter fragments with blunt ends were produced by PCR-amplification with Vent® DNA Polymerase (New England Biolabs, Beverly, MA) using forward primers CKXP1 and CKXP2 (both with the XbaI site), respectively, and a common reverse primer DSC2-GSP1 (Fig. 1B). The resulting products were digested with XbaI and subcloned into the XbaI/SmaI site of pBI101 to form pBI-CP1 and pBI-CP2, respectively.
The 1.4 kb deletion fragment was obtained by digestion of the resulting 2.2 kb PCR fragments with SalI, and subsequently cloned into the SaII/SmaI site to generate pBI-CP3.
The 0.92 kb promoter fragment with blunt end amplified by PCR amplification using CKXP4 (Fig. 1B) with a BamHI site and DSC2-GSP1 was digested with BamHI and ligated into BamHI/SmaI site of pBI101 to create pBI-CP5.
The 1.14, 0.79, 0.63, and 0.39 kb promoter fragments were amplified using forward primers CKXP3, CKXP5, CKXP6, and CKXP7, respectively, and a common reverse primer DSC2-GSP1 (Fig. 1B). These resulting PCR products were cloned into pGEM-T Easy Vector (Promega) to introduce new SalI sites at the 5' ends of these deletion fragments. Using these pGEM-T Easy Vectors containing distinct DSCKX1 promoter fragments as templates, the corresponding promoter fragments containing proper restriction sites were PCR-amplified with SP6 and DSC2-GSP1 primers. These PCR products were cut with SalI and cloned into the SalI/SmaI site of pBI101 to generate constructs pBI-CP4, pBI-CP6, pBI-CP7, and pBI-CP8, respectively.
All promoter constructs were sequenced to eliminate possible PCR-introduced mutations.
Plant transformation
GUS fluorometric activity for each DSCKX1 promoter construct described above was assayed using the transient orchid transformation system. Plasmids containing DSCKX1 promoter deletion–GUS fusions were isolated by the Wizard® Plus SV Minipreps DNA Purification System (Promega) and coated on gold particles by co-precipitation as described by Klein et al. (1987). For microprojectile bombardment, thin section explants (1 mm thickness) from PLBs were precultured for 3 d in liquid modified KC medium, and then evenly placed on a central core 2 cm in diameter on solid modified KC medium (Yu et al., 2000). These sections were bombarded using a Model Biolistic PDS-1000/He (Bio-Rad Laboratories) at 1350 psi helium gas pressure of the projectile; 60 mm Hg of partial vaccum; 0.5 mg of DNA-coated gold particle; and 9 cm distance from the stopping plate to the surface of the treated tissues. After bombardment, thin section explants were cultured for 2 d on solid modified KC medium with or without 25 µM BA and then collected for transient fluorometric GUS assays (Yu et al., 2002).
Agrobacterium tumefaciens strain LBA4404 carrying different promoter constructs was used to transform Arabidopsis via floral dip transformation as described in Clough and Bent (1998). The presence and integrity of transgenes were examined by Southern blot analysis as described previously (Yang et al., 2002).
Analysis of GUS activity
Fluorometric assays of GUS activity were performed according to Jefferson et al. (1987). Protein concentration in the supernatant was determined by the method of Bradford (1976). Histochemical detection of GUS activity was performed as described by Jefferson (1987) and Topping and Lindsey (1997). Tissues for GUS staining were incubated in staining solution (50 mM sodium phosphate, pH 7.0, 10 mM EDTA, 2 mM 5-bromo-4-chloro-3-indoyl glucuronide, 1 mM potassium ferricyanide, and 1 mM potassium ferrocyanide) at 37 °C overnight. After incubation, stained tissues were cleared of chlorophyll in an ethanol series.
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Results and discussion |
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TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
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A cytokinin oxidase gene DSCKX1 in Dendrobium orchid has previously been identified and characterized (Yang et al., 2002). In this study, the investigation on the regulation of DSCKX1 expression was extended by the identification of the possible regulatory elements in its upstream regulatory region.
Isolation and sequence analysis of the DSCKX1 5'-upstream region
The orchid DSCKX1 5'-upstream regulatory region was isolated by a genomic DNA walking strategy using a series of gene-specific primers derived from the DSCKX1 cDNA sequence and its upstream promoter region. Three successive PCR-based DNA walkings eventually resulted in the identification and cloning of the 3724 bp DSCKX1 putative promoter fragment as shown in Fig. 1A. 5'-RACE was employed to determine the transcription start site of the DSCKX1 gene. The two successive PCRs after reverse transcription of total RNA extracted from BA-treated leaves amplified a single major band of 230 bp in length (data not shown). Analysis of this amplified fragment revealed that the 5' end of the product was the putative transcription start site (+1) that is 33 bp upstream of the initiation codon (Met) of the DSCKX1 gene (Fig. 1A).
A putative TATA box (TATAAAT) sequence was located at –30 to –24 relative to the transcription start site, which was consistent with the regular feature of eukaryotic promoters (Zhu et al., 1995; Joshi, 1987). A CAAT box (CAAT) was also present at –67 to –64.
Further analysis of the DSCKX1 putative regulatory region with TFSEARCH and PLACE programs revealed the presence of a number of putative cis-elements for certain important regulatory proteins in plant development. As shown in Fig. 1, two putative as-1 like TGACG motifs (Lam and Chua, 1989; Benfey and Chua, 1990) required for cytokinin responses are present in the DSCKX1 promoter regions (–2346 to –2342 and –1226 to –1222). Furthermore, three elements similar to the motif of AAGATTGATTGAG, which is involved in the cytokinin induction of hprA (Jin et al., 1998), were found in positions –2939 to –2927 (AAGTTTGATTAAG), –2636 to –2624 (AAAATTGAATCAG) and –507 to –495 (AAAATTGATTAAC) (Fig. 1A).
Analysis of the DSCKX1 putative promoter in transient orchid transformation system
As a first step towards the identification of cis-regulatory elements involved in the control of DSCKX1 gene expression, the putative promoter deletions have been analysed by transient assays in orchid thin section from PLBs. These derived 5'-deletion fragments were transcriptionally fused to the same site upstream of GUS reporter gene to generate the constructs from pBI-CP1 to pBI-CP8 (Fig. 2). GUS activity in PLBs expressing the full-length DSCKX1::GUS fusion construct (construct pBI-CP1) was set at 100% and used to define the activities of other deletions. Removal of the sequence to –2189 (construct pBI-CP2) caused an obvious increase (about 3-fold) in the level of DSCKX1::GUS expression, while the deletion of an additional 785 bp of putative regulatory fragment (construct pBI-CP3) resulted in a significant decrease in promoter activity. Further deletion down to –1135 (construct pBI-CP4) did not significantly affect the promoter activity and subsequent deletions abolished promoter activity completely.
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These results indicated two regions of 5' upstream region contributing to promoter activity. The region from –3724 to –2189 appears negatively to control DSCKX1::GUS expression, whereas the region from –2189 to –1500 may contain enhancer(s) that promote DSCKX1::GUS expresion.
Since DSCKX1 was induced by cytokinin (Yang et al., 2002), the GUS expression of a series of deletion constructs was investigated further to determine the regulatory promoter sequence in response to cytokinin in this transient transformation system. As shown in Fig. 2, the DSCKX1::GUS fusion (pBI-CP1) showed an approximately 2.5-fold higher level of GUS activity in PLBs treated with BA (25 µM) than those in PLBs without BA treatment. Compared with this high level of BA-induced DSCKX1::GUS expression, plants transformed with both pBI-CP2 and pBI-CP3 constructs, exhibited relatively low changes of promoter activity after BA treatment. In particular, pBI-CP4 showed almost unchanged promoter activity under BA treatment. These results suggest that the sequence from –3724 to –404, especially from position –3724 to –2189 is important in the BA-regulation of cytokinin oxidase.
Analysis of DSCKX1 putative promoter in stable Arabidopsis transformation system
The authors are interested in defining the cis-elements responsible for the temporal and spatial expression of the DSCKX1 gene. Although Agrobacterium-mediated transformation of Dendrobium orchid is successful as described previously ((Yu et al., 2001), it is relatively time- and labour-consuming. Therefore, the well-established Arabidopsis transformation system was used instead. This has successfully been used to analyse heterologous promoter sequences (Haritatos et al., 2000; Moreno-Fonseca and Covarrubias, 2001) in order to locate putative cis-acting elements in the DSCKX1 promoter regions.
A series of 5' deletions were transcriptionally fused to the GUS reporter gene, and the resulting constructs were introduced into Arabidopsis by Agrobacterium-mediated transformation. The control Arabidopsis plants were transformed with a promoterless GUS construct (pBI101) or a 35S::GUS construct (pBI-121). Six to 15 independent transformants for each construct were obtained and examined for GUS activity by both quantitative and histochemical assays.
To investigate the effects of 5' putative promoter deletions on tissue-specific patterns, transformants harbouring different constructs were histochemically stained for GUS activity (Table 2). In plants transformed with pBI-CP1 construct, higher DSCKX1::GUS expression was observed in leaves, followed by roots and stems. Deletion to –2189 (pBI-CP2) produced similar but much stronger GUS expression patterns compared with pBI-CP1. Interestingly, deletion of sequences upstream of position –1404 (pBI-CP3) abolished DSCKX1 promoter activity in both roots and stems, and further deletion down to –794 caused the loss of promoter activity in leaves. These results suggested that the sequences responsible for root- and stem-specific DSCKX1 expression are present upstream of –1404. Analysis of the GUS staining patterns in stable Arabidopsis transformants also suggested that the regulation of DSCKX1 expression in leaves may depend on three discrete promoter regions. One region maps to the position –3724 to –2189, where negative cis-acting element(s) may exist to inhibit the DSCKX1 activity. The other two regions located at –2189 to –1404 and –923 to –794 could contain one or more positive regulatory element(s) required for promotion of DSCKX1 expression in leaves.
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The temporal and spatial distribution of DSCKX1 promoter-driven gene expression was investigated in several organs of the 30-d-old plants carrying pBI-CP2 construct, whose GUS expression pattern is stronger than pBI-CP1. Although there was some degree of variability in the level of expression among independent lines, the pattern of GUS expression driven by the DSCKX1 promoter was consistent with a high expression in rosette leaves and low in roots and stems (Fig. 3; Table 2). On average, GUS activity in rosette leaves (3100 pmol 4-MU mg–1 protein min–1) was 1.6-fold and 2.0-fold higher than that in roots and stems, respectively.
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To study the promoter activity during plant development, the transformants containing pBI-CP2 construct were examined for DSCKX1::GUS expression at different developmental stages (Figs 2, 4). The DSCKX1::GUS expression was not detectable in seedlings, including leaves, petioles and roots, until they were 20-d-old (Fig. 3). The relatively high DSCKX1::GUS expression was found in old leaves, followed by roots and stem. The expressions in these tissues became much stronger as the seedling developed. However, very low levels of GUS activity were detected in flowers and siliques.
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The GUS histochemical assay was performed in order to have a more detailed localization of the DSCKX1::GUS expression (Fig. 4). The putative DSCKX1 promoter affected GUS expression in specific tissues of transgenic Arabidopsis. As shown in Fig. 4, the X-Gluc staining was found predominantly in the veins and petioles of the rosette leaves and slightly in roots. In stems, the GUS activity was mainly located in the nodes and pedicels of flowers. However, the young leaves showed no detectable GUS staining.
Identification of DSCKX1 promoter regions containing cis-acting element(s) in response to cytokinins
Since it was found that DSCKX1 was induced by cytokinin application in orchid, an attempt was made, therefore, to study the cis-acting elements in response to BA induction using DSCKX1 promoter deletions. Six independent transgenic Arabidopsis plants from each construct, including the control vectors, were examined. For pBI-CP1 construct, GUS expression in leaves could be strongly induced when leaf discs were placed on MS medium containing BA (Fig. 5). Leaves containing pBI-CP2 and pBI-CP3 constructs showed lower induction in the presence of BA. However, the inducibility was lost in the transgenic plants containing the construct pBI-CP4, suggesting that some sequences, mainly in the region between –3724 and –1135 may be implicated in the regulation of the DSCKX1 gene in response to cytokinin. Furthermore, promoter activity of pBI-CP1 construct was enhanced with the increase of exogenous BA concentration, and reached the maximum at 25 µM (Fig. 5A). Additionally, significant induction of promoter activity in leaves was observed with the treatment of 25 µM BA after 12 h and continuously increased until 36 h after treatment (Fig. 5B). However, without BA treatment, the promoter activity in leaves did not change significantly during the period of incubation (data not shown).
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In addition to BA, kinetin, isopentenyl adenine, isopentenyl adenosine, zeatin, and zeatin riboside could also induce GUS expression, while adenine did not have the same effect (Fig. 6). These results are comparable to previous studies of the effect of different cytokinins on DSCKX1 expression at both mRNA and protein levels (Yang et al., 2002).
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The DSCKX1::GUS fusions showed cytokinin-inducible expression in both transgenic orchid and Arabidopsis. It is noteworthy that the cytokinin-inducible expression of DSCKX1::GUS in Arabidopsis was similar to the cytokinin-inducible expression of DSCKX1::GUS in orchid, with respect to parameters such as BA concentrations and the preferential induction by various cytokinins. In the DSCKX1 putative promoter region, there are two putative motifs homologous to cytokinin-responsive elements present between –3724 and –2189; one at position –2939 to –2927 and the other at –2636 to –2624. Moreover, two as-1 like motifs (TGACG) are present within the region from –2346 to –2342 and –1226 to –1222, respectively. Taken together, these results suggest that these cytokinin-responsive elements and as-1 like motifs may play important roles in regulating the cytokinin-inducible expression of the DSCKX1 gene.
In summary, the present study has demonstrated that the DSCKX1 promoter sequences contain specific cis-acting elements required for the spatial and temporal control of DSCKX1 expression during plant development. In particular, the promoter also contains the important regulatory regions essential for cytokinin-inducible transcription of the DSCKX1 gene. The mechanism of transcriptional regulation of DSCKX1 will be elucidated by the identification of the related trans-acting factors.
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Acknowledgement |
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This research was supported by research grant no. R-154-000-095-112 from the National University of Singapore.
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