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The regulation of Korean radish cationic peroxidase promoter by a low ratio of cytokinin to auxin
Plant Science 162 (2002) 345– 353 www.elsevier.com/locate/plantsci
The regulation of Korean radish cationic peroxidase promoter by a low ratio of cytokinin to auxin Dong Ju Lee a, Sung Soo Kim b, Soung Soo Kim b,* a b
MSU-DOE Plant Research Laboratory, Michigan State Uni6ersity, E. Lansing, MI 48824, USA Department of Biochemistry, College of Science, Yonsei Uni6ersity, Seoul 120 -749, South Korea Received 10 April 2001; received in revised form 23 August 2001; accepted 5 September 2001
Abstract Regulation of the Korean radish cationic peroxidase (KRCP) promoter by auxins– cytokinins are described in this study. Various fragments of the KRCP promoter have been cloned and transcriptionally fused to the GUS gene to transform tobacco BY-2 cells, arabidopsis, and tobacco plants. Cytokinins decreased the dose- and time-dependent GUS expression of the pBK12 construct in the BY-2 cells. In contrast, the GUS expression of the pBK12 construct was significantly induced by a low ratio of cytokinin to auxin, although the GUS expression was not observed in any organ of the transgenic arabidopsis and tobacco plants at any stage of normal development under no hormone treatment. The GUS activities of the pBK12 construct driven by a low ratio of cytokinin to auxin were over 400- and 58-fold higher in the leaves and stems, respectively, than in those of the untreated arabidopsis plants. The induced GUS staining was mainly localized in the leaves and stems treated with the low ratio of cytokinin to auxin. These results suggested that the promoter activity of the KRCP gene be up-regulated by the low ratio of cytokinin to auxin. The KRCP promoter exhibited a higher degree of specificity to a low ratio of cytokinin to auxin rather than to either auxin or cytokinin alone in tobacco and arabidopsis plants. To date this study provides the first evidence that the combination of cytokinin and auxin takes part more in the regulation of the activity of KRCP promoter than in each hormone alone. © 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: KRCP; Auxin; Cytokinin; Peroxidase; Synergism
1. Introduction The plant peroxidase isozymes (EC 1.11.1.7) are thought to be involved in various cellular functions, including the catabolism of auxin [7], production and removal of hydrogen peroxide [27], polymerization of phenolics into lignin [20], wound healing [6], defense against pathogen attacks [19], and somatic embryogenesis [4]. The correlation between plant organogenesis and the expression of different isoperoxidases in tobacco suggests that individual isoperoxidases may have a specific role in plant growth, development, and differentiation [15]. Klotz et al. [18] reported that the promoter activity of tobacco anionic peroxidase was tissue- and * Corresponding author. Tel./fax: + 82-2-2123-2698. E-mail address: [email protected] (S.S. Kim).
organ-specific and was highly regulated in all organs throughout development. The promoter of a basic isoperoxidase gene (prxC2) from horseradish (Armoracia rusticana) also directed the GUS expression highly in transgenic tobacco leaves, stems, and roots [14]. In addition, the promoter of Arabidopsis ATP A2 peroxidase directed the GUS gene expression in lignified tissues of transgenic plants [32]. The most widely used type of growth regulators are auxin and cytokinin. Auxins are considered to be a key phytohormone that is involved in controlling the rate of cell elongation, cell division in suspension culture cells and tissues, vascular differentiation, the initiation of lateral roots, and the control of lateral shooting [29]. On the other hand, cytokinins are known to be involved in the promotion of plant cell growth, regulation of the formation of vegetative buds from apical domi-
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nance, and the delay of senescence in vitro [36,38] or in vivo [26]. Cytokinins also inhibit the auxin-induced cell elongation of sunflower and soybean hypocotyl segments [5,40]. Auxin and cytokinin regulate plant development, controlling many aspects of plant growth and differentiation through a multitude of their interactions. Although the genetic investigations of the interaction between auxin and cytokinin signaling have been limited, the balance of auxins and cytokinins controls the formation of callus, shoots, and roots in vitro [36]. The mode of their interactions can be synergistic, antagonistic, or additive and is dependent on the type of tissue and the plant species [3]. When combined with auxin, cytokinin triggers a variety of phenomena: differentiation and development of cells, organs and whole plants [1]. Generally, a high ratio of cytokinin to auxin gives the best results of shoot induction [31] and it is well known that calli on low-cytokinin medium do not regenerate [34]. Based on our previous studies of peroxidase activity and isoenzyme pattern change during shoot initiation conditions of tobacco callus (N. tabacum cv Virginia 115), each isoperoxidase or groups of isoperoxidases function in different capacities. Therefore, the overall concentration of peroxidases may not be an important factor in the shoot-initiating process [16]. Several putative regulatory elements have been identified in the KRCP promoter. For instance, as-2 boxes (GATAN-GATA), which are known to confer leaf and shoot expression in tobacco, are conserved at − 280, − 40, and + 218 site [21]. SBF-1 core binding site (GGTTAA), with which SBF-1 nuclear factor interacts, functions as a transcriptional silencer which is involved in the organ-specific expression in plant development [22]. In addition, this site is conserved 148 bp downstream from the transcription start site in the KRCP promoter, and there are several putative GT elements at − 347 and + 364 sites, which the GT-1 factor, closely related to SBF-1, binds [23]. A putative gibberellin response element (GARE, TAACAAA) is located downstream of +151 relative to the transcriptional initiation site in the Korean radish cationic peroxidase gene [9]. We have shown earlier that the 5% upstream regions of a Korean radish cationic peroxidase gene are regulated in transgenic BYK12 cells by gibberellic acid and abscisic acid [24,25]. The changes of the activities of the Korean radish isoperoxidases by GA3 and/or abscisic acid (ABA) are similar to the result of GA3-inducible and/or ABA-repressible GUS expression mediated by the KRCP promoter in transgenic BYK12 cells. In this present paper, we show that a ratio of cytokinin to auxin causes effects on the activity of the KRCP promoter and on the tissue- and organ-specific expression pattern.
2. Materials and methods
2.1. Plasmid construction The genomic clone, named prxK1, has been described earlier in Ref. [33]. PCR amplifications of the KRCP promoter from positions − 471, − 241, − 132 to + 698 were performed with 1, 2, 3 and 6 primers, which were synthesized, based on the nucleotide sequences of prxK1. The sequence of the upstream primers 1 (5%-GCGCTCTAGAAGCTTTCCTCTTCGTTTAC-3%), 2 (5%-GCGCTCTAGATTTCTATTACTGTATGTTAA-3%), and 3 (5%-GCGCTCTAGATTGCCAGGTCTCGATCAATT-3%) contains a XbaI restriction enzyme site. The sequence of the downstream primer 6 (5%-GCGCACCCGGGTCGAAGACTTCATATAGTT-3%) contains an XmaI restriction enzyme site to create the new restriction enzyme site. PCR products were isolated, cloned into a pCR-Script™ Cam SK( +) cloning vector (Stratagene, La Jolla, Calif.), digested with XbaI and XmaI enzymes, and subcloned into a XbaI/XmaI polylinker site of the pBI101 vector, a Ti plasmid vector (Clontech, Palo Alto, Calif.). The new constructs (pBK12, pBK09, and pBK08) were verified by the nucleotide sequence and used to transform the Agrobacterium tumefaciens strain LBA4404 cells by the electroporation method [28].
2.2. Plant materials Tobacco suspension cells (Nicotiana tabacum L. cv BY-2) were maintained in a modified LS liquid medium containing 0.2 mg/ml of 2,4-D at 25 °C with shaking at 100 rpm on a gyrotary shaker and subcultured every 2 weeks with a 5% inoculum [1,35]. BY-2 cells were transformed by co-culture with the A. tumefaciens transfected with the pBK12, pBK09, pBK08, or pBI101 plasmids as described in Ref. [1]. The kanamycin-resistant transformant cells could be detected for 1 month.
2.3. Preparation of transgenic arabidopsis and tobacco plants Root transformation of arabidopsis with agrobacterium was carried out according to the method described earlier in Refs. [12,39] with some modifications. The regenerated plantlets which produced roots were washed with tap water and transferred to soil to set seeds. Seeds (T1) of the transformed arabidopsis plants were grown on GM plates containing 0.05 g/l kanamycin. The seedlings were scored for their ability to survive on kanamycin after 7–10 days. Seeds (T2) were harvested after 4–5 weeks.
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The chimeric plasmids (pBK12, pBK09, and pBK08) were stably inserted into the genomes of N. tabacum cv. Samsun by leaf disc method using Agrobacterium-mediated transformation [11]. When shoots appeared, individual shoots were cut clearly to separate sibling shoots. When roots appeared from the base of the shoots, the plantlets were removed, washed free of agar, and planted in soil. The transformed tobacco plants were grown in a greenhouse. For each construct, more than eight transgenic plant lines were analyzed in the T2 generation of arabidopsis and T0 generation of tobacco.
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These paraffin blocks were cut into 10-mm thick sections and mounted on slides. After removal of paraffin using xylene, these tissue sections were rehydrated using graded ethanol series and were counterstained with 0.5% safranine O for 10 s. These double-stained sections were photographed with a microscope on Kodak Gold 100 film. The total soluble protein was determined by the method of Bradford using BSA as a standard [2].
3. Results
2.4. Phytohormone treatment of transgenic tobacco cells, arabidopsis and tobacco plants Several concentrations of cytokinins were added to 8 ml tobacco cells of the pBK12 construct 7– 10 days after the subculture was performed. Cells were then cultured for 4 h or various time intervals for the GUS assay. Four-week-old arabidopsis plants (T2 generation), which had been grown in sterile condition, were treated in MSO medium (MS basal medium, B5 vitamins, 0.5 g/l 2-[N-Morpholino]ethane sulfonic acid (MES), 2% sucrose, pH 5.7) without hormone and with the given hormone under a cycle of 16 h of light and 8 h of dark at 28 °C for 4 days, respectively. The MSA medium comprised the MSO medium containing a low ratio of cytokinin to auxin (0.5 mg/l 2,4-D and 0.05 mg/l kinetin), which was widely used as a suspension culture medium of arabidopsis callus. The leaves of the transgenic tobacco plants between 8 and 12 cm long were excised and surface-sterilized. The midvein was removed. The leaf discs (1.0× 1.0 cm) were incubated on MS media (MS basal medium, B5 vitamins, 3% sucrose, pH 5.7) or the hormone-containing media {0.2 mg/l 2,4-D, 1 mg/l 2,4-D, and 1.87 mg/l p-chlorophenoxy acetic acid (pCPA) plus 0.442 mg/l 2,4-D plus 0.11 mg/l kinetin} at 25 °C for 8 days.
2.5. GUS assay and histochemical staining GUS assays were performed and evaluated using 4-methylumbelliferyl b-D-glucuronic acid (4-MUG) [8]. The histochemical staining was performed by incubating arabidopsis plants with X-gluc solution at 37 °C as described by Gallagher [8]. Chlorophyll was removed by incubating plants in 100% ethanol at room temperature. After staining with X-gluc, tissue samples were fixed in FAA solution (5% formalin, 5% glacial acetic acid and 90% EtOH) at room temperature for 1 day under infiltration, and then rinsed five times for 20 min in 100 mM phosphate buffer (pH 7.2) and twice in distilled water for 10 min. After dehydration using a series of graded alcohol for 20 min each and clearing with xylene, samples were embedded in hard paraffin.
3.1. Phytohormone effects in the BY-2 cells In order to understand the mechanisms of the KRCP gene expression, we have examined the response of the KRCP promoter to cytokinin and auxin. The KRCP promoter construct (pBK12) had a high expression level of GUS (about 2–3 nmol 4-MU/min/mg soluble protein) in the BY-2 cells [24], which were originally maintained in LS medium containing 0.2 mg/l of 2,4-D [30]. Dose dependence of the GUS gene expression was studied 4 h after treatment with various cytokinins in the BY-2 cells. Changes in GUS expression were recorded as a percent of control GUS activity (zero time after added cytokinins). Fig. 2(A) shows that all cytokinins can prohibit the expression of the GUS gene at non-toxic levels (from 0.5 to 2 mg/l) within 4 h of hormone treatment, though they prohibited it modestly from 10 to 30%. Natural cytokinins of zeatin and 2-iP have more ability to decrease the GUS activity than the synthetic cytokinins such as BAP and kinetin. There is no significant difference of concentration effect on this inhibition. Time-course experiments were carried out to explore the mode of GUS-suppression by cytokinins. As shown in Fig. 2(B), the GUS expression decreased slowly and consistently from 4 to 24 h after the application of 8 mg/l of 2-iP, a naturally occurring cytokinin, reaching eventually from 20% to almost 30% inhibition. A similar time-dependent inhibition pattern was also observed after treatment with 8 mg/l of BAP, a synthetic cytokinin.
3.2. GUS expression of the transgenic arabidopsis and tobacco plants Although the BY-2 cell line has several advantageous properties such as high rate of growth, high efficiency of transformation, and uniformity as a suspension, this cell line has lost the ability to regenerate into plantlets [30]. In this regard, we decided to examine the activity of the KRCP promoter in transgenic plants. Arabidopsis and tobacco were stably transformed with the gene chimera, the same plasmid used to transform tobacco
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BY-2 cells. Twelve arabidopsis plants of the pBK12 construct (T0 generation) were regenerated from individual calli, which axenically cultured on media containing kanamycin for positive selection. We chose the T2 generation of transgenic arabidopsis seeds exhibiting 100% kanamycin resistance. Heritable transmission of the KRCP promoter was confirmed by the genomic DNA analysis of T2 generation plants selected on MS media containing kanamycin by a PCR method (data not shown). The GUS expression was examined in whole plants at different developmental stages. There was no GUS expression in any organ of seedlings and mature transgenic arabidopsis, both assayed with 4MUG and stained with X-gluc (Fig. 7(A) and (C)). Thirty individual tobacco plants were obtained for the pBK12 construct, 20 plants for the pBK09 construct, and 18 plants for the pBK08 construct. Regenerated plants were grown vegetatively in a greenhouse. The GUS expression in whole transgenic plants grown under the same condition was examined. The GUS activities of the pBK12, the pBK09, and the pBK08 construct were barely detectable in all tissues of the tobacco plants as in arabidopsis plants (data not shown). The leaves, stems, and roots in all the tobacco plants were also not stained using X-gluc. Both nontransformed tobacco plants and transgenic tobacco plants for promoterless pBI101 plasmid did not express the GUS activity in any tissue, including reproductive organs (data not shown). This mode of the GUS expression in both arabidopsis and tobacco plants is very different from those of the tobacco anionic peroxidase promoter [18], horseradish cationic peroxidase promoter [14], and arabidopsis ATP A2 peroxidase promoter [32]. The GUS expression directed by 3 kb of the tobacco anionic peroxidase promoter was observed in leaves, stems, petioles, roots, flowers, and fruits of the transgenic tobacco plants. Also, high levels of GUS activity were observed in leaves, stems, and roots of transgenic tobacco plants that contained 1 kb of the 5% flanking region of horse radish peroxidase prxC2 fused to the GUS gene. Transgenic plants with a 709 bp promoter fragment of ATP A2 peroxidase gene expressed the marker enzymes in the vascular tissue of hypocotyl and leaves, roots, and other tissues.
3.3. Phytohormone effects in transgenic arabidopsis and tobacco plants The promoter activity of the pBK12 construct was compared between arabidopsis and tobacco plants after treatment with 2,4-D or kinetin. Several plants of the arabidopsis were incubated with phytohormones for 4 days. The GUS activities in the leaves and stems were induced from 100 to 250 pmol 4-MU/min/mg soluble protein, when treated with 0.05– 2.5 mg/l of kinetin or
2,4-D (Fig. 3). The leaves and stems in this case of induction were stained dimly to blue with X-gluc (data not shown). On the contrary, higher concentration of kinetin (2.5 mg/l) could not induce the GUS activity. When the leaf discs of the three mature tobacco plants of the pBK12 construct (12–2, 12–4, and 12–6 lines) were treated with 0.2– 1 mg/l of 2,4-D alone, the GUS activities were induced slightly from approximately 27 to 60 pmol 4-MU/min/mg soluble protein (Fig. 4).
3.4. The inducti6e effects of a low ratio of cytokinin to auxin in arabidopsis and tobacco plants To investigate the effect of a low ratio of cytokinin to auxin on the activity of the KRCP promoter, the tobacco leaf discs of the pBK12 construct (12– 2, 12–4, and 12–6 lines) were mounted on solid media with phytohormones. The GUS activity of leaf discs treated with a low ratio of cytokinin to auxin {1.87 mg/l p-chlorophenoxy acetic acid (pCPA) plus 0.442 mg/l 2,4-D plus 0.11 mg/l kinetin} was induced more than ten times compared with the set of untreated plants (control) (Fig. 4). It was also increased more than three times than that of leaf discs treated with 0.2–1 mg/l of 2,4-D alone. And other low ratios of cytokinin to auxin (0.2 mM kinetin plus 2 mM 2,4-D, and 0.5 mM kinetin plus 4 mM 2,4-D) also induced similarly the GUS activities of the pBK12, pBK09, and pBK08 constructs (data not shown). When the leaf discs of the pBK09 and pBK08 constructs were treated with the abovementioned low ratio of cytokinin to auxin, similar inducing results were observed, although the overall GUS activities were less induced compared with those of the pBK12 construct (Fig. 5). Several arabidopsis plants were incubated in a low ratio of cytokinin to auxin (0.05 mg/l kinetin plus 0.5 mg/l 2,4-D) for 4 days. The GUS expression was induced greatly to 15361 and 4921 pmol 4-MU/min/mg soluble protein in the leaves and stems, respectively (Fig. 6). This means that the combination of both 2,4-D and a low level of kinetin have a strong synergistic effect on the activation of the KRCP promoter, especially in the pBK12 construct. We reported earlier that ABA decreased the inductive GUS expression of the pBK12 construct by gibberellic acid in BY-2 cells [24]. When 10 mM ABA was also added to the MSA medium, ABA completely inhibited the inducing effect by the low ratio of cytokinin to auxin on the GUS activity of the pBK12 construct in arabidopsis.
3.5. Histochemical localization of the GUS expression To characterize the regulation of the pBK12 construct specifically, GUS staining was localized histochemically after the arabidopsis plants were treated
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with a low ratio of cytokinin to auxin or 2,4-D alone. Increased GUS activity was directly proportional to the intensity of an insoluble indigo-blue product. All procedures of plant growth and hormone treatment were carried out under sterile conditions in order to assure that the contamination of glucuronidase activity by some bacteria, fungi, and other organisms was excluded. These transgenic plants treated with auxin alone gave much less intense GUS staining (data not shown) than those treated with a low ratio of cytokinin to auxin. The GUS activity was mainly localized in the leaves and stems when plants were treated with a low ratio of cytokinin to auxin (Fig. 7(B), (D) and (E)). The vascular tissue system of petioles and leaves, and epidermal cells and cortical cells of stems were stained strong against a clear background. The epidermal cells of stems showed the most intense staining (blue color) among the various stained cells. Lignin was stained red with safranine O. The intensity of the inductive GUS activity was relatively weak in lignified tissues where safranine O stained red intensively (Fig. 7(E)). Therefore, the KRCP gene product may not be related to the formation of lignin which the tobacco anionic peroxidase and arabidopsis ATP A2 peroxidase had been suggested to catalyze.
4. Discussion Several fragments of the KRCP promoter have been cloned and fused to the GUS gene to transform N. tabacum cv BY-2 cells, A. thaliana ecotype Wassilewakila, and N. tabacum cv Samsun (Fig. 1). This KRCP promoter construct had a high expression level of GUS gene (about 2–3 nmol 4-MU/min/mg soluble protein) in the BY-2 cells transformed with the pBK12 construct [24]. The GUS expression of the pBK12 construct was not observed in any organ of the arabidopsis (Fig. 7(A) and (C)) and that of the pBK12, pBK09, and pBK08 constructs in tobacco plants (data not shown) at any
Fig. 2. Effect of cytokinins on the GUS expression of the pBK12 construct in BY-2 cells. (A) Measurement of the GUS activity 4 h after the addition of cytokinins (0.5 – 2 mg/l). (B) The time-course effect of 8 mg/l of 2-iP and BAP on the GUS expression over a period of 24 h. The values have been shown relative to the GUS activity of non-treated cells that was given a value of 100. The error bars represent standard error of the mean (n = 12). Fig. 1. Restriction maps and schematic constructions of the KRCP promoter/GUS fusion in the pBI101 plasmid. Primers (1, 2, 3, and 6) are indicated by arrows, which were used for PCR cloning. PCR amplifications of the Korean radish cationic peroxidase promoter from positions − 471 (pBK12), − 241 (pBK09), − 132 (pBK08) to + 698 were performed. The GUS marker enzyme is indicated by a solid line with an arrow. Nos-ter represents the nopaline synthase (nos) terminator.
stage of normal development. These observations indicate that the KRCP gene promoter contains transcriptionally a strong silencer element unlike the other peroxidase genes, and at least this silencing phenomenon is conserved between arabidopsis and tobacco
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plants. This silencing may be related with the putative three cis-elements, GGTTAA, (position at − 347, + 148, and + 364) with which a transcriptional silencer, such as the SBF-1 nuclear factor, is known to bind. The cis-element, with which the SBF-1 nuclear factor from bean interacts, functions as a transcriptional silencer that is involved in organ-specific expression in plant development [22]. We have examined the effect of auxin and cytokinin on the KRCP promoter activity. On the treatment of BY-2 cells with cytokinins, such as BAP, kinetin, zeatin, and 2-iP, the GUS activity decreased slightly and consistently in a dose- and time-dependent manner (Fig. 2(A) and (B)). In addition, a higher concentration of cytokinins than that of auxins is important to decrease the GUS expression in BY-2 cells. This suppression would be associated with phenomena triggered by the high level of cytokinins, such as the onset of plant regeneration [37]. Thus, the function of KRCP gene may be different from that of the tobacco anionic peroxidase gene which is not affected by the treatment of BAP [17]. Cytokinin is also well known to regulate the expression of plant peroxidase genes. The putative peroxidase (P17) accumulates in Petunia hybrida calli under an extremely low concentration of cytokinin [34].
Fig. 3. Effect of 2,4-D or kinetin on the GUS expression of the pBK12 construct in arabidopsis plants. Four-week-old arabidopsis plants grown under sterile condition were treated with given hormone for 4 days, respectively. Stems and leaves were used to measure the level of GUS expression. The error bars represent standard error of the mean (n = 8).
Fig. 4. Effect of hormones (0.2 mg/l 2,4-D, 1 mg/l 2,4-D, and 1.87 mg/l pCPA plus 0.442 mg/l 2,4-D plus 0.11 mg/l kinetin) on the GUS expression of the pBK12 construct in tobacco plants (12 – 2, 12 –4, and 12 – 6 lines). The inductive effect of a low ratio of cytokinin to auxin on the activity of the KRCP promoter is stronger than that of auxin alone. The leaf discs were surface-sterilized and incubated in solid medium for 8 days and were then assayed for the induced GUS activities with 4-MUG. The error bars represent standard error of the mean (n =8).
Also, the expression of petunia anionic peroxidase was reported to increase by callus-inducing stimulus [13]. Auxin or low levels of cytokinin alone showed an ability to induce the GUS activity of the pBK12 construct in the arabidopsis (Fig. 3). However, high levels of cytokinin did not activate the expression of the GUS gene. This means that high concentrations of cytokinin also repress the activity of the KRCP promoter in intact plants as it did in suspension cells. 2,4-D alone could slightly activate the GUS expression of the pBK12 construct in tobacco (Fig. 4). However, cytokinin alone could hardly induce the GUS expression (data not shown). Follow-up experiments confirmed that the cytokinin–auxin interaction has a pivotal role in inducing the KRCP promoter activity. The GUS expression of the pBK12 construct was greatly induced in the arabidopsis treated with a low ratio of cytokinin to auxin, which can induce callus formation (Fig. 6). The low ratio of cytokinin to auxin, which can induce callus of tobacco, had a stronger inducing effect on the GUS activity of the pBK12 construct than auxin alone in tobacco (Fig. 4) although auxin can also induce the tobacco callus. The low ratio of cytokinin to auxin induced the GUS activity of the pBK09 and pBK08 constructs in tobacco less than that of the pBK12 construct (Fig. 5). Therefore, these observations indicate that the low ratio of cytokinin to auxin acts in
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Fig. 5. Effect of a low ratio of cytokinin to auxin (1.87 mg/l pCPA plus 0.442 mg/l 2,4-D plus 0.11 mg/l kinetin) on the GUS expression of the pBK12, pBK09, and pBK08 constructs in different lines of tobacco plants. The low ratio of cytokinin to auxin induced the GUS activity of the pBK12 construct more than that of the pBK09 and pBK08 constructs. The leaf discs were surface-sterilized and incubated in hormone-containing medium for 8 days and then the GUS activities were assayed with 4-MUG. Three plants for each construct were chosen arbitrarily. The same pattern of graph represents the same plant. The error bars represent standard error of the mean (n =8).
concert with the increase of the KRCP promoter activity of the pBK12 construct in both arabidopsis and tobacco. Further analysis with the histochemical method revealed that the low ratio of cytokinin to auxin played a more important role in the induction of the GUS expression of the pBK12 construct than auxin or cytokinin alone in arabidopsis. The induced GUS staining was mainly localized in the leaves (Fig. 7(B)) and stems (Fig. 7(D) and (E)) treated with the low ratio of cytokinin to auxin. Taken together, analyses of the GUS activity apparently demonstrate that the low ratio of cytokinin to auxin can activate more transcriptionally the expression of the KRCP gene rather than each of the two hormones by itself. These experiments suggest that the KRCP promoter exhibits a higher degree of specificity to a low ratio of cytokinin to auxin rather than to either auxin or cytokinin alone in tobacco and arabidopsis plants. That is to say, the combined presence of cytokinin and auxin provided synergistic rather than additive effects. This synergistic phenomenon verified inversely that the GUS expression was suppressed by high concentrations of cytokinins, which cause plant regeneration, in BY-2 cells (Fig. 2(A) and (B)) and in arabidopsis plants (Fig. 3; 2.5 mg/l Kin). This mode is similar to the expression pattern of tobacco cationic peroxidases (C1, C2, and C14) that were known to be highly or newly expressed in a low ratio of cytokinin to
Fig. 6. Effect of a low ratio of cytokinin to auxin (0.05 mg/l kinetin and 0.5 mg/l 2,4-D, MSA medium) and a low ratio of cytokinin to auxin plus 10 mM abscisic acid (ABA) on the GUS expression of the pBK12 construct in arabidopsis. The GUS expression was induced greatly by a low ratio of cytokinin to auxin (MSA medium) in the leaves and stems, respectively. ABA inhibited completely the inducing effect by a low ratio of cytokinin to auxin on the GUS activity of the pBK12 construct. The error bars represent standard error of the mean (n =8).
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Fig. 7. Histochemical localization of the GUS expression in transgenic arabidopsis plants directed by the pBK12 construct. (A) untreated leaf (3.5 × ); (B) leaf treated with a low ratio of cytokinin to auxin; (C) untreated stem section (35 ×); (D) (30 × ) and (E) (120 × ), stem section treated with a low ratio of cytokinin to auxin. Samples of stems were cut into 10-mm sections, counterstained with safranine O, and viewed. The GUS expression was detected by incubating the plants in X-gluc substrate solution. Chlorophyll was removed by incubation in 70% ethanol at room temperature. Abbreviations: c, cortex; e, epithelium; p, pith; vs, vascular system.
auxin, but not usually expressed in a high ratio of cytokinin to auxin [15]. It was reported that treatment with both auxin and cytokinin led synergistically to higher expression levels of the GUS gene than with either of the hormones taken individually under the control of an Arabidopsis cdc2 promoter [10]. Skoog and Miller [36] demonstrated that the course of plant regeneration in culture could be determined by the ratio of cytokinin to auxin. ABA completely inhibited the effect of a cytokinin –auxin interaction on the induction of the GUS expression of the pBK12 construct in arabidopsis (Fig. 6). ABA alone did not induce the GUS expression of any pBK construct in arabidopsis (data not shown). This result suggests that there may be putative ABA-response cis-element(s), which reduces severely the inductive activity of the KRCP promoter by a low ratio of cytokinin to auxin and by gibberellic acid as shown earlier in Ref. [24]. The treatment of cycloheximide (CHX) also completely inhibited the
inductive GUS activity in arabidopsis plants which suggests that the ongoing protein synthesis is required for induction by a low ratio of cytokinin to auxin, or in other aspects of the process (data not shown). The interactions of auxin and cytokinin are very complex throughout plant growth, development, differentiation and senescence. Although the molecular mechanisms of auxin–cytokinin interactions are not well known, these are thought to include the mutual control of auxin and cytokinin metabolisms, interactions in the control of gene expression, and post-transcriptional interactions [3]. The KRCP promoter is able to determine both tissue- and cell-specific control of GUS expression when induced by the low ratio of cytokinin to auxin (Fig. 7(B), (D), and (E)). Considering the fact that separate sequence motifs are responsible for the interaction between the different regulatory signals, the dissection of the upstream cis elements in the KRCP gene may allow a definitive functional assignment to different sequence motifs. Through comparison studies among the promoter fragments, it appears that the putative low ratio of cytokinin to auxin-associated response element is located between −471 and − 241 in KRCP promoter (Fig. 5). Since the activity of the KRCP promoter (1.2 kb) was highly induced by the low ratio of cytokinin to auxin in both arabidopsis and tobacco plants, evolutionary linkage of the hormonal signaling pathway of the low ratio of cytokinin to auxin may be conserved among these species. It would be worthwhile to examine whether the KRCP promoter also works in Korean radish. In vivo and in vitro analyses of transgenic suspension culture cells and plants provide an opportunity to examine the molecular mechanisms underlying the developmental plasticity of Korean radish cationic peroxidase promoter. Particularly the KRCP promoter may be used as a marker for the investigation of cytokinin–auxin interaction in plants. Gel shift assay, finger printing, and site-directed mutagenesis for potential regulatory elements await future work.
Acknowledgements We gratefully acknowledge Jae Heung Ko, Department of Biology, Yonsei University, for his advice on plant transformation. We also would like to thank Dr E.S. Kim of Konkuk University, South Korea, for his expert analysis of the histochemical data, and Kyoung Nam Ko and Bum Suk Cho for histochemical experiments. This work was supported by the Korea Ministry of Education (Project No. BSRI-98-4420) and partially supported by Brain Korea 2001.
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The regulation of Korean radish cationic peroxidase promoter by a low ratio of cytokinin to auxin Dong Ju Lee a, Sung Soo Kim b, Soung Soo Kim b,* a b
MSU-DOE Plant Research Laboratory, Michigan State Uni6ersity, E. Lansing, MI 48824, USA Department of Biochemistry, College of Science, Yonsei Uni6ersity, Seoul 120 -749, South Korea Received 10 April 2001; received in revised form 23 August 2001; accepted 5 September 2001
Abstract Regulation of the Korean radish cationic peroxidase (KRCP) promoter by auxins– cytokinins are described in this study. Various fragments of the KRCP promoter have been cloned and transcriptionally fused to the GUS gene to transform tobacco BY-2 cells, arabidopsis, and tobacco plants. Cytokinins decreased the dose- and time-dependent GUS expression of the pBK12 construct in the BY-2 cells. In contrast, the GUS expression of the pBK12 construct was significantly induced by a low ratio of cytokinin to auxin, although the GUS expression was not observed in any organ of the transgenic arabidopsis and tobacco plants at any stage of normal development under no hormone treatment. The GUS activities of the pBK12 construct driven by a low ratio of cytokinin to auxin were over 400- and 58-fold higher in the leaves and stems, respectively, than in those of the untreated arabidopsis plants. The induced GUS staining was mainly localized in the leaves and stems treated with the low ratio of cytokinin to auxin. These results suggested that the promoter activity of the KRCP gene be up-regulated by the low ratio of cytokinin to auxin. The KRCP promoter exhibited a higher degree of specificity to a low ratio of cytokinin to auxin rather than to either auxin or cytokinin alone in tobacco and arabidopsis plants. To date this study provides the first evidence that the combination of cytokinin and auxin takes part more in the regulation of the activity of KRCP promoter than in each hormone alone. © 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: KRCP; Auxin; Cytokinin; Peroxidase; Synergism
1. Introduction The plant peroxidase isozymes (EC 1.11.1.7) are thought to be involved in various cellular functions, including the catabolism of auxin [7], production and removal of hydrogen peroxide [27], polymerization of phenolics into lignin [20], wound healing [6], defense against pathogen attacks [19], and somatic embryogenesis [4]. The correlation between plant organogenesis and the expression of different isoperoxidases in tobacco suggests that individual isoperoxidases may have a specific role in plant growth, development, and differentiation [15]. Klotz et al. [18] reported that the promoter activity of tobacco anionic peroxidase was tissue- and * Corresponding author. Tel./fax: + 82-2-2123-2698. E-mail address: [email protected] (S.S. Kim).
organ-specific and was highly regulated in all organs throughout development. The promoter of a basic isoperoxidase gene (prxC2) from horseradish (Armoracia rusticana) also directed the GUS expression highly in transgenic tobacco leaves, stems, and roots [14]. In addition, the promoter of Arabidopsis ATP A2 peroxidase directed the GUS gene expression in lignified tissues of transgenic plants [32]. The most widely used type of growth regulators are auxin and cytokinin. Auxins are considered to be a key phytohormone that is involved in controlling the rate of cell elongation, cell division in suspension culture cells and tissues, vascular differentiation, the initiation of lateral roots, and the control of lateral shooting [29]. On the other hand, cytokinins are known to be involved in the promotion of plant cell growth, regulation of the formation of vegetative buds from apical domi-
0168-9452/02/$ - see front matter © 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S0168-9452(01)00564-7
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nance, and the delay of senescence in vitro [36,38] or in vivo [26]. Cytokinins also inhibit the auxin-induced cell elongation of sunflower and soybean hypocotyl segments [5,40]. Auxin and cytokinin regulate plant development, controlling many aspects of plant growth and differentiation through a multitude of their interactions. Although the genetic investigations of the interaction between auxin and cytokinin signaling have been limited, the balance of auxins and cytokinins controls the formation of callus, shoots, and roots in vitro [36]. The mode of their interactions can be synergistic, antagonistic, or additive and is dependent on the type of tissue and the plant species [3]. When combined with auxin, cytokinin triggers a variety of phenomena: differentiation and development of cells, organs and whole plants [1]. Generally, a high ratio of cytokinin to auxin gives the best results of shoot induction [31] and it is well known that calli on low-cytokinin medium do not regenerate [34]. Based on our previous studies of peroxidase activity and isoenzyme pattern change during shoot initiation conditions of tobacco callus (N. tabacum cv Virginia 115), each isoperoxidase or groups of isoperoxidases function in different capacities. Therefore, the overall concentration of peroxidases may not be an important factor in the shoot-initiating process [16]. Several putative regulatory elements have been identified in the KRCP promoter. For instance, as-2 boxes (GATAN-GATA), which are known to confer leaf and shoot expression in tobacco, are conserved at − 280, − 40, and + 218 site [21]. SBF-1 core binding site (GGTTAA), with which SBF-1 nuclear factor interacts, functions as a transcriptional silencer which is involved in the organ-specific expression in plant development [22]. In addition, this site is conserved 148 bp downstream from the transcription start site in the KRCP promoter, and there are several putative GT elements at − 347 and + 364 sites, which the GT-1 factor, closely related to SBF-1, binds [23]. A putative gibberellin response element (GARE, TAACAAA) is located downstream of +151 relative to the transcriptional initiation site in the Korean radish cationic peroxidase gene [9]. We have shown earlier that the 5% upstream regions of a Korean radish cationic peroxidase gene are regulated in transgenic BYK12 cells by gibberellic acid and abscisic acid [24,25]. The changes of the activities of the Korean radish isoperoxidases by GA3 and/or abscisic acid (ABA) are similar to the result of GA3-inducible and/or ABA-repressible GUS expression mediated by the KRCP promoter in transgenic BYK12 cells. In this present paper, we show that a ratio of cytokinin to auxin causes effects on the activity of the KRCP promoter and on the tissue- and organ-specific expression pattern.
2. Materials and methods
2.1. Plasmid construction The genomic clone, named prxK1, has been described earlier in Ref. [33]. PCR amplifications of the KRCP promoter from positions − 471, − 241, − 132 to + 698 were performed with 1, 2, 3 and 6 primers, which were synthesized, based on the nucleotide sequences of prxK1. The sequence of the upstream primers 1 (5%-GCGCTCTAGAAGCTTTCCTCTTCGTTTAC-3%), 2 (5%-GCGCTCTAGATTTCTATTACTGTATGTTAA-3%), and 3 (5%-GCGCTCTAGATTGCCAGGTCTCGATCAATT-3%) contains a XbaI restriction enzyme site. The sequence of the downstream primer 6 (5%-GCGCACCCGGGTCGAAGACTTCATATAGTT-3%) contains an XmaI restriction enzyme site to create the new restriction enzyme site. PCR products were isolated, cloned into a pCR-Script™ Cam SK( +) cloning vector (Stratagene, La Jolla, Calif.), digested with XbaI and XmaI enzymes, and subcloned into a XbaI/XmaI polylinker site of the pBI101 vector, a Ti plasmid vector (Clontech, Palo Alto, Calif.). The new constructs (pBK12, pBK09, and pBK08) were verified by the nucleotide sequence and used to transform the Agrobacterium tumefaciens strain LBA4404 cells by the electroporation method [28].
2.2. Plant materials Tobacco suspension cells (Nicotiana tabacum L. cv BY-2) were maintained in a modified LS liquid medium containing 0.2 mg/ml of 2,4-D at 25 °C with shaking at 100 rpm on a gyrotary shaker and subcultured every 2 weeks with a 5% inoculum [1,35]. BY-2 cells were transformed by co-culture with the A. tumefaciens transfected with the pBK12, pBK09, pBK08, or pBI101 plasmids as described in Ref. [1]. The kanamycin-resistant transformant cells could be detected for 1 month.
2.3. Preparation of transgenic arabidopsis and tobacco plants Root transformation of arabidopsis with agrobacterium was carried out according to the method described earlier in Refs. [12,39] with some modifications. The regenerated plantlets which produced roots were washed with tap water and transferred to soil to set seeds. Seeds (T1) of the transformed arabidopsis plants were grown on GM plates containing 0.05 g/l kanamycin. The seedlings were scored for their ability to survive on kanamycin after 7–10 days. Seeds (T2) were harvested after 4–5 weeks.
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The chimeric plasmids (pBK12, pBK09, and pBK08) were stably inserted into the genomes of N. tabacum cv. Samsun by leaf disc method using Agrobacterium-mediated transformation [11]. When shoots appeared, individual shoots were cut clearly to separate sibling shoots. When roots appeared from the base of the shoots, the plantlets were removed, washed free of agar, and planted in soil. The transformed tobacco plants were grown in a greenhouse. For each construct, more than eight transgenic plant lines were analyzed in the T2 generation of arabidopsis and T0 generation of tobacco.
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These paraffin blocks were cut into 10-mm thick sections and mounted on slides. After removal of paraffin using xylene, these tissue sections were rehydrated using graded ethanol series and were counterstained with 0.5% safranine O for 10 s. These double-stained sections were photographed with a microscope on Kodak Gold 100 film. The total soluble protein was determined by the method of Bradford using BSA as a standard [2].
3. Results
2.4. Phytohormone treatment of transgenic tobacco cells, arabidopsis and tobacco plants Several concentrations of cytokinins were added to 8 ml tobacco cells of the pBK12 construct 7– 10 days after the subculture was performed. Cells were then cultured for 4 h or various time intervals for the GUS assay. Four-week-old arabidopsis plants (T2 generation), which had been grown in sterile condition, were treated in MSO medium (MS basal medium, B5 vitamins, 0.5 g/l 2-[N-Morpholino]ethane sulfonic acid (MES), 2% sucrose, pH 5.7) without hormone and with the given hormone under a cycle of 16 h of light and 8 h of dark at 28 °C for 4 days, respectively. The MSA medium comprised the MSO medium containing a low ratio of cytokinin to auxin (0.5 mg/l 2,4-D and 0.05 mg/l kinetin), which was widely used as a suspension culture medium of arabidopsis callus. The leaves of the transgenic tobacco plants between 8 and 12 cm long were excised and surface-sterilized. The midvein was removed. The leaf discs (1.0× 1.0 cm) were incubated on MS media (MS basal medium, B5 vitamins, 3% sucrose, pH 5.7) or the hormone-containing media {0.2 mg/l 2,4-D, 1 mg/l 2,4-D, and 1.87 mg/l p-chlorophenoxy acetic acid (pCPA) plus 0.442 mg/l 2,4-D plus 0.11 mg/l kinetin} at 25 °C for 8 days.
2.5. GUS assay and histochemical staining GUS assays were performed and evaluated using 4-methylumbelliferyl b-D-glucuronic acid (4-MUG) [8]. The histochemical staining was performed by incubating arabidopsis plants with X-gluc solution at 37 °C as described by Gallagher [8]. Chlorophyll was removed by incubating plants in 100% ethanol at room temperature. After staining with X-gluc, tissue samples were fixed in FAA solution (5% formalin, 5% glacial acetic acid and 90% EtOH) at room temperature for 1 day under infiltration, and then rinsed five times for 20 min in 100 mM phosphate buffer (pH 7.2) and twice in distilled water for 10 min. After dehydration using a series of graded alcohol for 20 min each and clearing with xylene, samples were embedded in hard paraffin.
3.1. Phytohormone effects in the BY-2 cells In order to understand the mechanisms of the KRCP gene expression, we have examined the response of the KRCP promoter to cytokinin and auxin. The KRCP promoter construct (pBK12) had a high expression level of GUS (about 2–3 nmol 4-MU/min/mg soluble protein) in the BY-2 cells [24], which were originally maintained in LS medium containing 0.2 mg/l of 2,4-D [30]. Dose dependence of the GUS gene expression was studied 4 h after treatment with various cytokinins in the BY-2 cells. Changes in GUS expression were recorded as a percent of control GUS activity (zero time after added cytokinins). Fig. 2(A) shows that all cytokinins can prohibit the expression of the GUS gene at non-toxic levels (from 0.5 to 2 mg/l) within 4 h of hormone treatment, though they prohibited it modestly from 10 to 30%. Natural cytokinins of zeatin and 2-iP have more ability to decrease the GUS activity than the synthetic cytokinins such as BAP and kinetin. There is no significant difference of concentration effect on this inhibition. Time-course experiments were carried out to explore the mode of GUS-suppression by cytokinins. As shown in Fig. 2(B), the GUS expression decreased slowly and consistently from 4 to 24 h after the application of 8 mg/l of 2-iP, a naturally occurring cytokinin, reaching eventually from 20% to almost 30% inhibition. A similar time-dependent inhibition pattern was also observed after treatment with 8 mg/l of BAP, a synthetic cytokinin.
3.2. GUS expression of the transgenic arabidopsis and tobacco plants Although the BY-2 cell line has several advantageous properties such as high rate of growth, high efficiency of transformation, and uniformity as a suspension, this cell line has lost the ability to regenerate into plantlets [30]. In this regard, we decided to examine the activity of the KRCP promoter in transgenic plants. Arabidopsis and tobacco were stably transformed with the gene chimera, the same plasmid used to transform tobacco
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BY-2 cells. Twelve arabidopsis plants of the pBK12 construct (T0 generation) were regenerated from individual calli, which axenically cultured on media containing kanamycin for positive selection. We chose the T2 generation of transgenic arabidopsis seeds exhibiting 100% kanamycin resistance. Heritable transmission of the KRCP promoter was confirmed by the genomic DNA analysis of T2 generation plants selected on MS media containing kanamycin by a PCR method (data not shown). The GUS expression was examined in whole plants at different developmental stages. There was no GUS expression in any organ of seedlings and mature transgenic arabidopsis, both assayed with 4MUG and stained with X-gluc (Fig. 7(A) and (C)). Thirty individual tobacco plants were obtained for the pBK12 construct, 20 plants for the pBK09 construct, and 18 plants for the pBK08 construct. Regenerated plants were grown vegetatively in a greenhouse. The GUS expression in whole transgenic plants grown under the same condition was examined. The GUS activities of the pBK12, the pBK09, and the pBK08 construct were barely detectable in all tissues of the tobacco plants as in arabidopsis plants (data not shown). The leaves, stems, and roots in all the tobacco plants were also not stained using X-gluc. Both nontransformed tobacco plants and transgenic tobacco plants for promoterless pBI101 plasmid did not express the GUS activity in any tissue, including reproductive organs (data not shown). This mode of the GUS expression in both arabidopsis and tobacco plants is very different from those of the tobacco anionic peroxidase promoter [18], horseradish cationic peroxidase promoter [14], and arabidopsis ATP A2 peroxidase promoter [32]. The GUS expression directed by 3 kb of the tobacco anionic peroxidase promoter was observed in leaves, stems, petioles, roots, flowers, and fruits of the transgenic tobacco plants. Also, high levels of GUS activity were observed in leaves, stems, and roots of transgenic tobacco plants that contained 1 kb of the 5% flanking region of horse radish peroxidase prxC2 fused to the GUS gene. Transgenic plants with a 709 bp promoter fragment of ATP A2 peroxidase gene expressed the marker enzymes in the vascular tissue of hypocotyl and leaves, roots, and other tissues.
3.3. Phytohormone effects in transgenic arabidopsis and tobacco plants The promoter activity of the pBK12 construct was compared between arabidopsis and tobacco plants after treatment with 2,4-D or kinetin. Several plants of the arabidopsis were incubated with phytohormones for 4 days. The GUS activities in the leaves and stems were induced from 100 to 250 pmol 4-MU/min/mg soluble protein, when treated with 0.05– 2.5 mg/l of kinetin or
2,4-D (Fig. 3). The leaves and stems in this case of induction were stained dimly to blue with X-gluc (data not shown). On the contrary, higher concentration of kinetin (2.5 mg/l) could not induce the GUS activity. When the leaf discs of the three mature tobacco plants of the pBK12 construct (12–2, 12–4, and 12–6 lines) were treated with 0.2– 1 mg/l of 2,4-D alone, the GUS activities were induced slightly from approximately 27 to 60 pmol 4-MU/min/mg soluble protein (Fig. 4).
3.4. The inducti6e effects of a low ratio of cytokinin to auxin in arabidopsis and tobacco plants To investigate the effect of a low ratio of cytokinin to auxin on the activity of the KRCP promoter, the tobacco leaf discs of the pBK12 construct (12– 2, 12–4, and 12–6 lines) were mounted on solid media with phytohormones. The GUS activity of leaf discs treated with a low ratio of cytokinin to auxin {1.87 mg/l p-chlorophenoxy acetic acid (pCPA) plus 0.442 mg/l 2,4-D plus 0.11 mg/l kinetin} was induced more than ten times compared with the set of untreated plants (control) (Fig. 4). It was also increased more than three times than that of leaf discs treated with 0.2–1 mg/l of 2,4-D alone. And other low ratios of cytokinin to auxin (0.2 mM kinetin plus 2 mM 2,4-D, and 0.5 mM kinetin plus 4 mM 2,4-D) also induced similarly the GUS activities of the pBK12, pBK09, and pBK08 constructs (data not shown). When the leaf discs of the pBK09 and pBK08 constructs were treated with the abovementioned low ratio of cytokinin to auxin, similar inducing results were observed, although the overall GUS activities were less induced compared with those of the pBK12 construct (Fig. 5). Several arabidopsis plants were incubated in a low ratio of cytokinin to auxin (0.05 mg/l kinetin plus 0.5 mg/l 2,4-D) for 4 days. The GUS expression was induced greatly to 15361 and 4921 pmol 4-MU/min/mg soluble protein in the leaves and stems, respectively (Fig. 6). This means that the combination of both 2,4-D and a low level of kinetin have a strong synergistic effect on the activation of the KRCP promoter, especially in the pBK12 construct. We reported earlier that ABA decreased the inductive GUS expression of the pBK12 construct by gibberellic acid in BY-2 cells [24]. When 10 mM ABA was also added to the MSA medium, ABA completely inhibited the inducing effect by the low ratio of cytokinin to auxin on the GUS activity of the pBK12 construct in arabidopsis.
3.5. Histochemical localization of the GUS expression To characterize the regulation of the pBK12 construct specifically, GUS staining was localized histochemically after the arabidopsis plants were treated
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with a low ratio of cytokinin to auxin or 2,4-D alone. Increased GUS activity was directly proportional to the intensity of an insoluble indigo-blue product. All procedures of plant growth and hormone treatment were carried out under sterile conditions in order to assure that the contamination of glucuronidase activity by some bacteria, fungi, and other organisms was excluded. These transgenic plants treated with auxin alone gave much less intense GUS staining (data not shown) than those treated with a low ratio of cytokinin to auxin. The GUS activity was mainly localized in the leaves and stems when plants were treated with a low ratio of cytokinin to auxin (Fig. 7(B), (D) and (E)). The vascular tissue system of petioles and leaves, and epidermal cells and cortical cells of stems were stained strong against a clear background. The epidermal cells of stems showed the most intense staining (blue color) among the various stained cells. Lignin was stained red with safranine O. The intensity of the inductive GUS activity was relatively weak in lignified tissues where safranine O stained red intensively (Fig. 7(E)). Therefore, the KRCP gene product may not be related to the formation of lignin which the tobacco anionic peroxidase and arabidopsis ATP A2 peroxidase had been suggested to catalyze.
4. Discussion Several fragments of the KRCP promoter have been cloned and fused to the GUS gene to transform N. tabacum cv BY-2 cells, A. thaliana ecotype Wassilewakila, and N. tabacum cv Samsun (Fig. 1). This KRCP promoter construct had a high expression level of GUS gene (about 2–3 nmol 4-MU/min/mg soluble protein) in the BY-2 cells transformed with the pBK12 construct [24]. The GUS expression of the pBK12 construct was not observed in any organ of the arabidopsis (Fig. 7(A) and (C)) and that of the pBK12, pBK09, and pBK08 constructs in tobacco plants (data not shown) at any
Fig. 2. Effect of cytokinins on the GUS expression of the pBK12 construct in BY-2 cells. (A) Measurement of the GUS activity 4 h after the addition of cytokinins (0.5 – 2 mg/l). (B) The time-course effect of 8 mg/l of 2-iP and BAP on the GUS expression over a period of 24 h. The values have been shown relative to the GUS activity of non-treated cells that was given a value of 100. The error bars represent standard error of the mean (n = 12). Fig. 1. Restriction maps and schematic constructions of the KRCP promoter/GUS fusion in the pBI101 plasmid. Primers (1, 2, 3, and 6) are indicated by arrows, which were used for PCR cloning. PCR amplifications of the Korean radish cationic peroxidase promoter from positions − 471 (pBK12), − 241 (pBK09), − 132 (pBK08) to + 698 were performed. The GUS marker enzyme is indicated by a solid line with an arrow. Nos-ter represents the nopaline synthase (nos) terminator.
stage of normal development. These observations indicate that the KRCP gene promoter contains transcriptionally a strong silencer element unlike the other peroxidase genes, and at least this silencing phenomenon is conserved between arabidopsis and tobacco
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plants. This silencing may be related with the putative three cis-elements, GGTTAA, (position at − 347, + 148, and + 364) with which a transcriptional silencer, such as the SBF-1 nuclear factor, is known to bind. The cis-element, with which the SBF-1 nuclear factor from bean interacts, functions as a transcriptional silencer that is involved in organ-specific expression in plant development [22]. We have examined the effect of auxin and cytokinin on the KRCP promoter activity. On the treatment of BY-2 cells with cytokinins, such as BAP, kinetin, zeatin, and 2-iP, the GUS activity decreased slightly and consistently in a dose- and time-dependent manner (Fig. 2(A) and (B)). In addition, a higher concentration of cytokinins than that of auxins is important to decrease the GUS expression in BY-2 cells. This suppression would be associated with phenomena triggered by the high level of cytokinins, such as the onset of plant regeneration [37]. Thus, the function of KRCP gene may be different from that of the tobacco anionic peroxidase gene which is not affected by the treatment of BAP [17]. Cytokinin is also well known to regulate the expression of plant peroxidase genes. The putative peroxidase (P17) accumulates in Petunia hybrida calli under an extremely low concentration of cytokinin [34].
Fig. 3. Effect of 2,4-D or kinetin on the GUS expression of the pBK12 construct in arabidopsis plants. Four-week-old arabidopsis plants grown under sterile condition were treated with given hormone for 4 days, respectively. Stems and leaves were used to measure the level of GUS expression. The error bars represent standard error of the mean (n = 8).
Fig. 4. Effect of hormones (0.2 mg/l 2,4-D, 1 mg/l 2,4-D, and 1.87 mg/l pCPA plus 0.442 mg/l 2,4-D plus 0.11 mg/l kinetin) on the GUS expression of the pBK12 construct in tobacco plants (12 – 2, 12 –4, and 12 – 6 lines). The inductive effect of a low ratio of cytokinin to auxin on the activity of the KRCP promoter is stronger than that of auxin alone. The leaf discs were surface-sterilized and incubated in solid medium for 8 days and were then assayed for the induced GUS activities with 4-MUG. The error bars represent standard error of the mean (n =8).
Also, the expression of petunia anionic peroxidase was reported to increase by callus-inducing stimulus [13]. Auxin or low levels of cytokinin alone showed an ability to induce the GUS activity of the pBK12 construct in the arabidopsis (Fig. 3). However, high levels of cytokinin did not activate the expression of the GUS gene. This means that high concentrations of cytokinin also repress the activity of the KRCP promoter in intact plants as it did in suspension cells. 2,4-D alone could slightly activate the GUS expression of the pBK12 construct in tobacco (Fig. 4). However, cytokinin alone could hardly induce the GUS expression (data not shown). Follow-up experiments confirmed that the cytokinin–auxin interaction has a pivotal role in inducing the KRCP promoter activity. The GUS expression of the pBK12 construct was greatly induced in the arabidopsis treated with a low ratio of cytokinin to auxin, which can induce callus formation (Fig. 6). The low ratio of cytokinin to auxin, which can induce callus of tobacco, had a stronger inducing effect on the GUS activity of the pBK12 construct than auxin alone in tobacco (Fig. 4) although auxin can also induce the tobacco callus. The low ratio of cytokinin to auxin induced the GUS activity of the pBK09 and pBK08 constructs in tobacco less than that of the pBK12 construct (Fig. 5). Therefore, these observations indicate that the low ratio of cytokinin to auxin acts in
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Fig. 5. Effect of a low ratio of cytokinin to auxin (1.87 mg/l pCPA plus 0.442 mg/l 2,4-D plus 0.11 mg/l kinetin) on the GUS expression of the pBK12, pBK09, and pBK08 constructs in different lines of tobacco plants. The low ratio of cytokinin to auxin induced the GUS activity of the pBK12 construct more than that of the pBK09 and pBK08 constructs. The leaf discs were surface-sterilized and incubated in hormone-containing medium for 8 days and then the GUS activities were assayed with 4-MUG. Three plants for each construct were chosen arbitrarily. The same pattern of graph represents the same plant. The error bars represent standard error of the mean (n =8).
concert with the increase of the KRCP promoter activity of the pBK12 construct in both arabidopsis and tobacco. Further analysis with the histochemical method revealed that the low ratio of cytokinin to auxin played a more important role in the induction of the GUS expression of the pBK12 construct than auxin or cytokinin alone in arabidopsis. The induced GUS staining was mainly localized in the leaves (Fig. 7(B)) and stems (Fig. 7(D) and (E)) treated with the low ratio of cytokinin to auxin. Taken together, analyses of the GUS activity apparently demonstrate that the low ratio of cytokinin to auxin can activate more transcriptionally the expression of the KRCP gene rather than each of the two hormones by itself. These experiments suggest that the KRCP promoter exhibits a higher degree of specificity to a low ratio of cytokinin to auxin rather than to either auxin or cytokinin alone in tobacco and arabidopsis plants. That is to say, the combined presence of cytokinin and auxin provided synergistic rather than additive effects. This synergistic phenomenon verified inversely that the GUS expression was suppressed by high concentrations of cytokinins, which cause plant regeneration, in BY-2 cells (Fig. 2(A) and (B)) and in arabidopsis plants (Fig. 3; 2.5 mg/l Kin). This mode is similar to the expression pattern of tobacco cationic peroxidases (C1, C2, and C14) that were known to be highly or newly expressed in a low ratio of cytokinin to
Fig. 6. Effect of a low ratio of cytokinin to auxin (0.05 mg/l kinetin and 0.5 mg/l 2,4-D, MSA medium) and a low ratio of cytokinin to auxin plus 10 mM abscisic acid (ABA) on the GUS expression of the pBK12 construct in arabidopsis. The GUS expression was induced greatly by a low ratio of cytokinin to auxin (MSA medium) in the leaves and stems, respectively. ABA inhibited completely the inducing effect by a low ratio of cytokinin to auxin on the GUS activity of the pBK12 construct. The error bars represent standard error of the mean (n =8).
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Fig. 7. Histochemical localization of the GUS expression in transgenic arabidopsis plants directed by the pBK12 construct. (A) untreated leaf (3.5 × ); (B) leaf treated with a low ratio of cytokinin to auxin; (C) untreated stem section (35 ×); (D) (30 × ) and (E) (120 × ), stem section treated with a low ratio of cytokinin to auxin. Samples of stems were cut into 10-mm sections, counterstained with safranine O, and viewed. The GUS expression was detected by incubating the plants in X-gluc substrate solution. Chlorophyll was removed by incubation in 70% ethanol at room temperature. Abbreviations: c, cortex; e, epithelium; p, pith; vs, vascular system.
auxin, but not usually expressed in a high ratio of cytokinin to auxin [15]. It was reported that treatment with both auxin and cytokinin led synergistically to higher expression levels of the GUS gene than with either of the hormones taken individually under the control of an Arabidopsis cdc2 promoter [10]. Skoog and Miller [36] demonstrated that the course of plant regeneration in culture could be determined by the ratio of cytokinin to auxin. ABA completely inhibited the effect of a cytokinin –auxin interaction on the induction of the GUS expression of the pBK12 construct in arabidopsis (Fig. 6). ABA alone did not induce the GUS expression of any pBK construct in arabidopsis (data not shown). This result suggests that there may be putative ABA-response cis-element(s), which reduces severely the inductive activity of the KRCP promoter by a low ratio of cytokinin to auxin and by gibberellic acid as shown earlier in Ref. [24]. The treatment of cycloheximide (CHX) also completely inhibited the
inductive GUS activity in arabidopsis plants which suggests that the ongoing protein synthesis is required for induction by a low ratio of cytokinin to auxin, or in other aspects of the process (data not shown). The interactions of auxin and cytokinin are very complex throughout plant growth, development, differentiation and senescence. Although the molecular mechanisms of auxin–cytokinin interactions are not well known, these are thought to include the mutual control of auxin and cytokinin metabolisms, interactions in the control of gene expression, and post-transcriptional interactions [3]. The KRCP promoter is able to determine both tissue- and cell-specific control of GUS expression when induced by the low ratio of cytokinin to auxin (Fig. 7(B), (D), and (E)). Considering the fact that separate sequence motifs are responsible for the interaction between the different regulatory signals, the dissection of the upstream cis elements in the KRCP gene may allow a definitive functional assignment to different sequence motifs. Through comparison studies among the promoter fragments, it appears that the putative low ratio of cytokinin to auxin-associated response element is located between −471 and − 241 in KRCP promoter (Fig. 5). Since the activity of the KRCP promoter (1.2 kb) was highly induced by the low ratio of cytokinin to auxin in both arabidopsis and tobacco plants, evolutionary linkage of the hormonal signaling pathway of the low ratio of cytokinin to auxin may be conserved among these species. It would be worthwhile to examine whether the KRCP promoter also works in Korean radish. In vivo and in vitro analyses of transgenic suspension culture cells and plants provide an opportunity to examine the molecular mechanisms underlying the developmental plasticity of Korean radish cationic peroxidase promoter. Particularly the KRCP promoter may be used as a marker for the investigation of cytokinin–auxin interaction in plants. Gel shift assay, finger printing, and site-directed mutagenesis for potential regulatory elements await future work.
Acknowledgements We gratefully acknowledge Jae Heung Ko, Department of Biology, Yonsei University, for his advice on plant transformation. We also would like to thank Dr E.S. Kim of Konkuk University, South Korea, for his expert analysis of the histochemical data, and Kyoung Nam Ko and Bum Suk Cho for histochemical experiments. This work was supported by the Korea Ministry of Education (Project No. BSRI-98-4420) and partially supported by Brain Korea 2001.
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