JXB Advance Access originally published online on February 21, 2005
Journal of Experimental Botany 2005 56(414):1165-1175; doi:10.1093/jxb/eri109
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RESEARCH PAPER |
Effects of cor15a-IPT gene expression on leaf senescence in transgenic Petuniaxhybrida and Dendranthemaxgrandiflorum
ková3
í Malbeck3
1University of Connecticut, Plant Science Department, Agricultural Biotechnology Laboratories, 1390 Storrs Rd, U-4163, Storrs, CT 06269-4163, USA
2Institute Biology and Soil Sciences, Far-Eastern Branch, Russian Academy of Sciences, Vladivostok, Russian Federation
3Institute of Experimental Botany, Academy of Sciences of the Czech Republic, Prague, Czech Republic
* To whom correspondence should be addressed. Fax: +1 860 486 0534. E-mail: Richard.McAvoy{at}uconn.edu
Received 13 August 2004; Accepted 20 December 2004
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Abstract |
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To prevent leaf senescence of young transplants or excised shoots during storage under dark and cold conditions, the cytokinin biosynthetic gene isopentenyl transferase (ipt) was placed under the control of a cold-inducible promoter cor15a from Arabidopsis thaliana and introduced into Petuniaxhybrida ‘Marco Polo Odyssey’ and Dendranthemaxgrandiflorum (chrysanthemum) ‘Iridon’. Transgenic cor15a-ipt petunia and chrysanthemum plants and excised leaves remained green and healthy during prolonged dark storage (4 weeks at 25 °C) after an initial exposure to a brief cold-induction period (4 °C for 72 h). However, cor15a-ipt chrysanthemum plants and excised leaves that were not exposed to a cold-induction period, senesced under the same dark storage conditions. Regardless of cold-induction treatment, leaves and plants of non-transformed plants senesced under prolonged dark storage. Analysis of ipt expression indicated a marked increase in gene expression in intact transgenic plants as well as in isolated transgenic leaves exposed to a short cold-induction treatment prior to dark storage. These changes correlated with elevated concentrations of cytokinins in transgenic leaves after cold treatment. Cor15a-ipt transgenic plants showed a normal phenotype when grown at 25 °C.
Key words: Cold-inducible promoter, cytokinins, ipt expression, leaf senescence, ornamental plants
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Introduction |
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Cytokinins regulate a number of growth and developmental processes in plants, such as stimulating cell division, maintaining plant vigour, delaying plant senescence and post-harvest and low-temperature-induced leaf yellowing (Gan and Amasino, 1997
). In practice, exogenous cytokinin applications are often not efficacious in commercial horticulture because they are expensive to use and are not readily assimilated (Hobbie et al., 1994). Barry et al. (1985) generated cytokinin overproducing petunia plants using the ipt gene cloned from Agrobacterium tumefaciens under the control of the CaMV 35S promoter. The ipt gene encodes the enzyme isopentenyl transferase, which catalyses the rate-limiting step in cytokinin biosynthesis (Akiyoshi et al., 1984). Excised leaves from cytokinin-overproducing ipt-transgenic plants displayed prolonged chlorophyll retention and delayed senescence (Li et al., 1992). The delayed senescence observed in intact and excised leaves from ipt-transformed plants demonstrates the potential utility of the ipt gene in extending the storage life of ornamental plants, green vegetables, and fruits. Unfortunately, most ipt-transgenic plants exhibit morphological abnormalities, restricting the potential for use in commercial production. This occurs because overproduction of cytokinins during crop growth interferes with normal development (Gan and Amasino, 1997). For example, elevated cytokinins resulted in a reduction in stature, the release from apical dominance, changes in vascular development, and, in many cases, an inhibition of root growth (Klee et al., 1987; Hobbie et al., 1994). To achieve better control of cytokinin overproduction, researchers fused the ipt gene to inducible promoters that were activated by stimuli such as heat (Medford et al., 1989; Smigocki, 1991; Ainley et al., 1993), wounding (Smigocki et al., 1993), or light (Thomas et al., 1995). However, in most cases, morphological changes in ipt-transgenic plants were observed even in the absence of an induction stimulus, suggesting leakage of the inducible system. Using the senescence-specific SAG12 promoter to drive ipt expression in tobacco, Gan and Amasino (1995) demonstrated that a specific developmental response could be elicited through more precise control of ipt expression. The SAG12 promoter activated ipt expression only at the onset of senescence, resulting in increased cytokinin concentrations in the senescing tissue and inhibition of the senescence process. Inhibition of leaf senescence by ipt expression led to the attenuation of the senescence-specific promoter, thus preventing cytokinin overproduction that would interfere with other aspects of development. In SAG12-ipt tobacco, leaf senescence was effectively controlled without other developmental abnormalities (Gan and Amasino, 1997). Subsequently, this strategy was successfully used in rice (Fu et al., 1998), cauliflower (Nguyen et al., 1998), petunia (Clark et al., 2004), and lettuce (McCabe et al., 2001). For example, leaf senescence was retarded in mature 60-d-old lettuce plants that exhibited normal morphology with no significant differences in head diameter or fresh weight of leaves and roots (McCabe et al., 2001).
In commercial horticulture, it is advantageous to be able to store whole live plants (seedlings) and excised shoots (cuttings) for extended periods of time without a loss of vitality. Plants and excised plant parts are typically stored under cool, dark conditions but the incidence of chilling injury and mortality increases with storage duration (Heins et al., 1995). At warmer temperatures, leaves senesce and overall plant quality deteriorates rapidly in dark storage. Controlled expression of ipt during dark storage, but not during normal crop production, could potentially increase and extend the storage tolerance of commercial crops without adversely affecting subsequent production in the field or greenhouse. In this study, an ipt fusion gene under the control of a cold-inducible promoter from the cor15a gene from Arabidopsis thaliana was constructed. Petunia and chrysanthemum plants were used to test the effects of this unique construct on storage tolerance, chlorophyll stability, and growth and development of transgenic plants.
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Materials and methods |
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Plasmid construction
Molecular cloning procedures were carried out as described by Sambrook et al. (1989)
. The ipt coding sequence used to construct the chimeric cor15a/ipt gene was derived from the pUC19 vector containing the ipt under the control of the 35S promoter. A 1 kb fragment of ipt-nos was obtained by double digestion of pUC19-35S-ipt-nos with EcoRI and SalI and cloned into the cloning site of the pBluescriptII KS vector. The 5' promoter and leader sequence from the cor15a gene (0.98 kb) was synthesized from genomic DNA of Arabidopsis thaliana by PCR reaction. The cloning sites for XhoI and SalI were added to the ends of the cor15a primers 5'-GGCTCGAGAGATCTTGTCCGTTGAATTT-3' and 5'-GGTCGACGAGAGAGATCTTTAAGATGT-3' for PCR. The cor15a fragment was subcloned into the pBluescriptII KS-ipt-nos vector by double digestion with XhoI and SalI to generate pBluescriptII KS with ipt under the control of the cor15a promoter. In the final step, the cor15-ipt-nos fragment was inserted into the multicloning site of the pBin19 vector by digesting pBluescriptII KS-cor15a-ipt-nos and pBin19 with KpnI and SmaI and then ligating the blunt ends. Thus, the binary pBin19 vector containing ipt under the control of the cor15a promoter was generated. The binary plasmid was transformed into Agrobacterium tumefaciens strain LBA 4404 by electroporation. The identity and accuracy of the cloned cor15a promoter sequence was confirmed by DNA sequence analysis (WM Keck Biotechnology Laboratory, Yale University, New Haven, CT).
Transformation and regeneration of transgenic plants
Petunia cv. Marco Polo Odyssey and chrysanthemum cv. Iridon were grown at 25 °C in the greenhouse in 3.8 l pots containing a peat-based substrate (Metro 510, Scotts Co., Marysville, Ohio). Plants were fertilized weekly with 400 mg l–1 N from a 20/4.3/16.6 N/P/K stock solution (Peter's 20-10-20, Scotts Co., Marysville OH). Plant shoots were cut at monthly intervals to induce new shoot growth. Leaf and stem tissue from young, newly developed shoots was used as explant tissue for plant transformation as follows.
Young, fully expanded petunia (cv. Marco Polo Odyssey) leaves were sterilized with 0.6% sodium hypochlorite (15–20 min) and then rinsed five times with sterile water. Stems from young, soft shoot tips of chrysanthemum plants (cv. Iridon) were washed for 60 s with 70% ethanol, rinsed three times with sterile water, and then sterilized in 0.3% sodium hypochlorite for 8 min before finally rinsing five times with sterile water. The bacterial suspension was cultured in LB medium supplemented with 50 mg l–1 kanamycin and 25 mg l–1 rifampicin. The suspension was incubated at 25 °C on a rotary shaker (220 rpm) until achieving an optical density of 0.4–0.7 (
600 nm). The suspension was then centrifuged and the pellet resuspended in a fresh liquid MS medium. Leaf explants of petunia or stem segments of chrysanthemum were soaked in the infection medium for 5 min, blotted dry and kept for 3 d in the dark at 22–25 °C on plates with MS medium containing 2 mg l–1 of N6-benzyladenine (BA), 0.01 mg l–1 of NAA for petunia explants or 0.225 mg l–1 of BA, 2 mg l–1 of IAA for chrysanthemum explants. After 2–3 d, explants were transferred to the respective selection media containing 50 mg l–1 of kanamycin (for selection) and 200 mg l–1 of timentin (to eliminate the Agrobacterium). Explants were transferred to fresh medium every 2–3 weeks, until shoots developed. Excised shoots were then transferred to phytohormone-free MS medium containing 50 mg l–1 of kanamycin and 100 mg l–1 of timentin until root induction was evident. Rooted explants were transferred to a peat-based medium (Metro 510, Scotts Co., Marysville, OH), and acclimated to the greenhouse environment.
Plant DNA extraction and polymerase chain reaction (PCR analysis)
Total DNA was isolated from leaf tissue using mini-prep kits (DNeasy Plant Mini Kit, Qiagen Inc., Valencia, CA, USA) and 250 ng of DNA was subjected to PCR reaction. The primers used to detect the cor15a-ipt locus were as follows: (i) for the full cor15a promoter (0.98 kb), forward primer 5'-GGCTCGAGAGATCTTGTCCGTTGAATTT-3' and reverse primer 5'-GGTCGACGAGAGAGATCTTTAAGATGT-3'; and for the 0.525 kb region of the ipt gene (ii) forward primer 5'-GGTCCAACTTGCACAGGAAAG-3' and reverse primer 5'-GGCTTGCCTACTGGAAGCTTA-3'. PCR amplification was performed using a thermocycler (GeneAmp PCR System 2700, Applied Biosystems, Inc., Foster City, CA, USA). Cycling conditions for both genes were 3 min at 94 °C and then 30 cycles of 1 min at 94 °C, 1 min at 54 °C and 1 min 30 s at 72 °C, followed by extension at 72 °C for 5 min. The reactions included 200–250 ng of DNA template, 0.2 mM of dNTPs, 0.5 µM of each primer, REDTaq PCR buffer and 1.5 U of REDTaq DNA polymerase (Sigma, St Louis, MO, USA). Finally, a 20 µl aliquot of PCR product was observed under UV after electrophoresis on a 1% agarose gel with ethidium bromide. A 1 kb DNA molecular marker (Gibco BRL) was used as a reference to determine DNA fragment size.
Southern hybridization
Total genomic DNA was isolated from transgenic plants using DNeasy Plant Maxi Kits (Qiagen Inc., Valencia, CA, USA) in accordance with the recommended protocol. Total genomic DNA from putative transgenic and non-transformed control plants (10 mg samples) was digested at 37° C overnight by double restriction with enzymes HindIII and EcoRI and cor15a-ipt fragment was released. Digested DNA from each line was separated through a 1% agarose gel prepared in TAE buffer, pH 8.5 (Sambrook et al., 1989) and fragments were transferred from agarose gel to a nylon membrane (Amersham, Chalfont St Giles, UK) and cross-linked to the membrane under UV irradiation. The ipt gene probe (a 0.525 kb fragment of the ipt gene) was prepared with a PCR DIG Probe synthesis kit (Roche Molecular Biochemicals, Indianapolis, IN) in accordance with the recommended protocol. The DNA fixed on membranes was prehybridized using a prehybridization solution at 68 °C for 3 h, and then hybridized with the probe at 68 °C overnight, and finally triple-washed with the post-hybridization solution at 65 °C in a hybridization oven (HB-2D, Techne Ltd., Duxford-Cambridge, UK). Solutions for sample hybridization, and pre- and post-hybridization, and the buffers for the following steps were prepared as previously reported by Mercier (1998). Membranes were washed for 5 min in 50 ml of maleate buffer (0.1 M maleic acid, 3.0 M of NaCl, pH 8.0) at room temperature and then incubated for 1 h in 50 ml of blocking solution (maleate buffer plus 0.5% blocking reagent: Roche Molecular Biochemicals, Indianapolis, IN). Membranes were then incubated for 30 min in 20 ml of blocking solution with anti-digoxigenin-AP, Fab fragments (Roche Molecular Biochemicals, Indianapolis, IN) diluted to 1:10 000 and then washed four times for 10 min in 50 ml of the maleate buffer. As a final step, membranes were equilibrated for 5 min. in 50 ml of substrate buffer (100 mM of TRIS–HCl, 100 mM of NaCl, 5 mM of MgCl2, pH 9.5) and then incubated at 37 °C for 10 min in 2 ml (sandwiched between two translucent plastic pages) of substrate buffer plus chemiluminescent substrate at a 1:100 dilution (CSPD, Roche Molecular Biochemicals, Indianapolis, IN). Membranes were exposed to autoradiographic film (Kodak X-Omart AR) for 4 h. X-ray films were developed with an automatic film processor.
Analysis of ipt expression in leaves of petunia and chrysanthemum
Using wild type and two cor15a-ipt-transgenic lines from both petunia and chrysanthemum, total RNA was isolated with TRI reagent (Molecular Research Center, Inc., Cincinnati, OH, USA) from samples that were frozen with liquid nitrogen and then ground in a mortar. For RT-PCR analysis, DNase treatment (DNA-freeTM, Ambion, Inc.) was used to eliminate DNA contamination from RNA samples, and then first-strand cDNA was synthesized from 1 µg of total RNA using First Strand Synthesis Kit RETROscriptTM (Ambion Inc. Austin, TX, USA) following the manufacturer's recommended protocol. For PCR, 0.5 µl of RT-mix was used in a final volume of 25 µl. PCR reaction for the ipt gene fragment was carried out as described above. PCR reaction products along with RT-mix and primers to 18S RNA were used as internal standards (QuantumRNATM 18S Internal Standards, Ambion Inc.). PCR products (10 µl) were run on a 1% agarose gel.
Senescence of excised leaves and shoots
To determine transgenic plant tolerance to prolonged dark storage, excised leaves from petunia and chrysanthemum were surface-sterilized with 0.6% sodium hypochlorite for 60 s, rinsed five times and then placed on moist filter paper in a 10 cm Petri dish. Leaves were selected from individual transgenic lines and non-transformed wild-type plants. Each Petri dish contained two excised leaves from both an individual transgenic plant line and a non-transformed wild-type plant. For petunia, five transgenic lines were tested. For chrysanthemum, two transgenic lines were tested. Identical plates were assembled for each transgenic line of petunia and chrysanthemum. The plates were either exposed to (i) a cold induction period (3 d at 4 °C in the dark) followed by continuous dark storage at 25 °C, or (ii) continuous dark storage at 25 °C without a prior cold induction treatment. Plates in dark storage were checked daily over a 28 d period for evidence of leaf senescence. Chlorophyll concentration was assayed prior to the start of each experiment and after significant loss of chlorophyll was detected in the non-transformed wild-type tissue. Each treatment combination was replicated in triplicate and the experiment was repeated three times. In separate experiments, whole shoots were excised from both wild-type plants and individual transgenic lines of petunia and chrysanthemum. Shoots from individual plants were bundled in groups of five and wrapped in a moist paper towel. Bundles from each transgenic line and the wild type were enclosed in a plastic bag and subjected to the same treatments and experimental protocol as previously described.
Quantification of chlorophyll
Specific chlorophyll concentration was determined, as follows. Wild-type and transgenic leaves, from each treatment plate in the previously described study, were blotted dry and weighed, and 100 mg of tissue from each sample placed in a 1.5 ml microcentrifuge tubes. The samples were resuspended in 80% acetone, ground with a disposable pestle, and incubated in the dark for 30 min. Total chlorophyll (Chl µg ml–1) was determined using absorbance at 645 nm and 663 nm according to the equation: 20.2 A645 + 8.02 A663 (Chory et al., 1994).
Morphological analysis of transgenic plants
The effect of the transgene on growth and development of chrysanthemums was determined in growth chamber studies. Thirty shoots from each transgenic cor15a-ipt chrysanthemum plant lines 9 and 12, and from the wild-type cultivar ‘Iridon’ were excised and rooted in deep 606-cell packs (Kord Products, Bramalea, Ontario, Canada) containing a Metro 510 (Scotts Co., Marysville, Ohio) peat-lite medium. After shoots were well rooted (3 weeks), the rooted cuttings were transferred to the growth chamber (EGC model S10, EGC, Chargrin Falls, OH) at 25/20 °C day/night (16 h at 300 µmol m–2 s–1). Plants were allowed to acclimate to the growth chamber conditions for 2 weeks and then 10 plants from each line were exposed to a cold induction period (3 d at 4°C) and then returned to the growth chamber while 10 plants from each line remained in the growth chamber without exposure to a cold induction period. In the growth chamber, plants were watered as needed and fertilized once per week with N at 5.3 mmol (75 mg l–1) from a 20/4.3/16.6 N/P/K stock solution (Peter's 20-10-20, Scotts Co., Marysville OH). After 6 weeks in the growth chamber five plants from each treatment were harvested and the following data recorded: shoot fresh weight (g), number of lateral shoots, lateral shoot length (cm), number of secondary shoots on each lateral, leaf area (cm2) on the uppermost lateral shoot, number of nodes on the uppermost lateral shoot, and total number of lateral shoots on the main stem. These parameters were used to calculate the average internode length and the average area per leaf on the uppermost lateral shoot. These data were used to determine difference in vegetative growth habit between transgenic and wild-type plants with or without exposure to a cold-induction period. In a separate study, the remaining 10 rooted chrysanthemum cuttings from each line were exposed to short-day conditions to induce flowering and the number of flower buds on each plant were recorded at anthesis.
Plants were arranged in a randomized complete block design with 10 replicated blocks. Statistical effects were determined using a 2-way analysis of variance with genetic line and cold-treatment as the main effects.
Cytokinin analysis
Cytokinins were extracted and purified according to the method of Dobrev and Kaminek (2002). Freeze-dried samples were homogenized with mortar and pestle in liquid nitrogen and extracted overnight with 10 ml methanol/water/formic acid (15/4/1, by vol., pH 
2.5, –20 °C). To each sample, 50 pmol of each of 12 deuterium labelled standards ([2H5]Z, [2H5]ZR, [2H5]Z7G, [2H5]Z9G, [2H5]ZOG, [2H5]ZROG, [2H6]iP, [2H6]iPA, [2H6]iP7G, [2H6]iP9G, [2H5]DHZ, [2H5]DHZR; products of Apex Organics, Honiton, UK) were added. The extract was passed through 2 ml Si-C18 columns (SepPak Plus, Waters, USA) to remove interfering lipophilic substances. After organic solvent evaporation in vacuo, the aqueous residue was applied to an Oasis MCX mixed mode (cation exchange and reverse-phase) column (150 mg, Waters, USA). Adsorbed cytokinins were eluted stepwise with 5 ml of 0.35 M ammonium in water (cytokinin nucleotides) and 0.35 M ammonium in 60% methanol (v/v) (cytokinin bases, ribosides, and glucosides). The eluted fractions were evaporated in vacuo. Nucleotide samples were dephosphorylated with acid phosphatase (0.6 U per sample) for 1 h at 37 °C. LC-MS analysis was performed using a Rheos 2000 HPLC gradient pump (Flux Instruments, Basel, Switzerland) and HIS PAL autosampler (CTC Analytics, Zwingen, Switzerland) coupled to an Ion Trap Mass Spectrometer Finnigan MAT LCQ-MSn equipped with an electrospray interface. Samples dissolved in 10% (v/v) acetonitrile (10 µl) were injected on a C18 column (Aqua 125A, 2 mm/250 mm/5 µm) and eluted with a linear gradient of B from 10% to 50% in 26 min (mobile phase: water (A), acetonitrile (B), and 0.001% (v/v) acetic acid in water (C) at a flow rate 0.2 ml min–1. Under these chromatographic conditions all analysed cytokinins were separated. Detection and quantification were carried out using a Finnigan LCQ operated in the positive ion, full-scan MS/MS mode using a multilevel calibration graph with deuterated cytokinins as internal standards. The levels of 19 different cytokinin derivatives were measured. The detection limit was calculated for each compound as 3.3 
/S, where is the standard deviation of the response and S the slope of the calibration curve. For each treatment, samples were collected from each of three independent plants and each sample was injected at least twice.
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Results |
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Gene construction and plant transformation
Transformation of petunia and chrysanthemum with the cor15a gene promoter-ipt gene (cor15a-ipt) construct resulted in more than 30 kanamycin-resistant putative transformants for each species. PCR and Southern hybridization analysis confirmed recombinant DNA integration into the genome of individual putative-transgenic petunia and chrysanthemum lines (Fig. 1). PCR amplification of both plasmid DNA and the genomic DNA from chrysanthemum lines produced the expected 0.98 kb fragment of the cor15a promoter (Fig. 1A) and the 0.52 kb fragment of the ipt gene (Fig. 1B). No amplification of DNA was detected in non-transgenic plants. Southern blot analysis of petunia genomic DNA revealed the integration of the ipt gene into the genome of several primary transformants, while no signal was detected in control plants (Fig. 1C). Transgenic plants of petunia were also confirmed by PCR reaction, and PCR positive lines of petunia and chrysanthemum were used for all subsequent experiments.
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Molecular analysis of transgenic plants expressing cor15a-ipt
Reverse transcription-PCR (RT-PCR) analysis was used to confirm ipt expression in transgenic lines in response to cold-induction signal. Total RNAs were extracted from the leaves of wild-type and selected transgenic lines of chrysanthemum (lines 9 and 12) and petunia (lines 7 and 9) that were grown under normal conditions or first exposed to a 3 d cold-induction (4 °C) treatment. RT-PCR analysis showed that the 0.52 kb ipt DNA fragment was amplified in both cor15a-ipt chrysanthemum (line 9) and cor15a-ipt petunia (line 7) exposed to the 4 °C treatment, but not in the same lines grown at the 25 °C and not exposed to the cold-induction treatment (Fig. 2). Similar results were obtained with line 9 of petunia and 12 line of chrysanthemum (data not shown). Wild-type plants showed no evidence of ipt gene expression regardless of temperature treatment. These data demonstrate that ipt expression in cor15a-ipt plants could be up-regulated with a cold-induction signal, but remained suppressed at normal growing temperatures.
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Leaf senescence
The leaf senescence response of chrysanthemum and petunia under long-term dark storage conditions differed markedly between cor15a-ipt and wild-type plants (Fig. 3). Overall, leaves from cold-induced cor15a-ipt plants remained green and healthy in prolonged dark storage while leaves from non-induced cor15a-ipt plants and from wild-type plants, regardless of cold-induction treatments, did not. Similar results were observed with excised leaves of both chrysanthemum and petunia, and excised shoots and whole intact plants of chrysanthemum. For example, excised leaves of both wild-type chrysanthemum and wild-type petunia showed a dramatic loss of chlorophyll and advanced tissue senescence after 28 d in continuous darkness at 25 °C. A pretreatment of cold-induction temperatures had little effect on the course of tissue senescence under these conditions. However, when excised leaves of cor15a-ipt chrysanthemum and cor15a-ipt petunia were exposed to a cold-induction treatment (4 °C for 3 d) and then stored in the dark for 28 d, the tissue showed little or no visible symptoms of chlorophyll loss or tissue senescence. Leaves of cor15a-ipt chrysanthemum and cor15a-ipt petunia that were not exposed to a cold-induction treatment prior to dark storage developed symptoms of chlorophyll loss or tissue senescence that approached those observed in wild-type leaves. With both excised shoots of cor15a-ipt chrysanthemum and whole intact cor15a-ipt chrysanthemum plants, a cold-induction treatment was required to produce the delayed onset of leaf senescence response under prolonged dark storage conditions.
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Quantitative analysis revealed that leaf chlorophyll concentrations in cor15a-ipt petunia lines and wild-type plants were similar under normal greenhouse growing conditions and showed a similar decline under prolonged dark storage conditions (Fig. 4). However, when plants were first exposed to a cold-induction treatment, the chlorophyll concentration in the cor15a-ipt lines remained at the level of normal grown plants even when exposed to prolonged dark storage. Cold induction had no beneficial effect on chlorophyll stability in the wild-type plants and chlorophyll concentrations showed a precipitous decline in response to dark storage. Experiments with excised leaves of wild-type and cor-15a-ipt chrysanthemum produced a similar response (data not shown).
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Changes in endogenous concentrations of cytokinins
Analysis of endogenous cytokinins, in freeze-dried petunia shoot tips from cor15a-ipt plants, revealed a dramatic increase after a cold-induction treatment compared to concentrations from wild-type plants (Table 1). Expression of the cor15a-ipt gene in petunia especially affected zeatin and dihydrozeatin type cytokinins. In cor15a-ipt plants, exposure to a cold-induction period (3.5 d at 4 °C) resulted in the increase of the physiologically active cytokinin trans-zeatin and its riboside (>4-fold and >18-fold, respectively), as well as of the storage cytokinins zeatin nucleotide (>10-fold increase), zeatin O-glucoside (5-fold increase) and the cytokinin deactivation products zeatin 7-glucoside (>10-fold increase) and zeatin-9-glucoside (>7-fold increase). The dihydrozeatin type cytokinins followed the same trend, but the increase was less dramatic. From the isopentenyladenine type cytokinins only the level of the active base (isopentenyladenine) was slightly increased during ipt expression at 4 °C. The concentration of isopentenyladenosine was considerably elevated under growth permissive conditions (25 °C) in both wild-type and transformed plants.
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In cor15a-ipt chrysanthemum plants, cold-induced ipt gene expression produced marked increases in both the storage cytokinin pool (P
0.05) and the pool of physiologically active cytokinins (P 0.05), but the total cytokinin pool (active, deactivated, and storage forms combined) was not substantially altered (Table 2). In more detail, trans-zeatin concentrations were similar in wild-type chrysanthemum, under both non-inducing (25 °C) and cold-inducing (4 °C) temperatures, and in non-induced cor15a-ipt plants (averaging 8.2 pmol g–1 DW). However, concentrations increased (P 0.05) in cor15a-ipt plants exposed to a prolonged period (14 d) at 4 °C and those exposed to a short cold induction period (3.5 d) followed by transfer to the 25 °C growth-permissive conditions for 3.5 d (averaging 16.8 pmol g–1 DW). However, 10.5 d after transfer to 25 °C the concentration of trans-zeatin in cold-induced cor15a-ipt plants decreased to non-induced concentrations. In cor15a-ipt plants exposed to 4 °C for either 7 d or for 3.5 d followed by 3.5 d at 25 °C, the concentration of trans-zeatin riboside (averaging 6.6 pmol g–1 DW) was measurably higher (P 0.05) than in non-induced cor15a-ipt plants and wild-type plants in both inductive and non-inductive conditions (averaging 1.5 pmol g–1 DW). The concentration of isopentenyladenine detected in non-induced wild-type, non-induced cor15a-ipt, and cold-induced wild-type plants (4.1 pmol g–1 DW), was higher (P 0.05) than the concentration found in cor15a-ipt plants exposed to cold for between 3.5 d and 14 d, or in plants exposed to cold for 3.5 d and then returned to 25 °C for 3.5 d (2.7 pmol g–1 DW). By contrast, the concentration of the corresponding riboside (iP7R) significantly increased (P 0.05) in cor15a-ipt plants induced in cold for 3.5 d and then returned to growth conditions for either 3.5 d or 10.5 d (13.1 pmol g–1 DW) compared to the average concentration found in cold-induced wild-type plants, non-induced wild-type plants and non-induced cor15a-ipt plants (7.1 pmol g–1 DW). The concentration of iP7R was dramatically lower (P 0.01) in cor15a-ipt plants after 3.5 d of cold-induction than when similar plants were transferred to growth conditions for 3.5 d or 10.5d. The concentration of dihydrozeatin was low in all plants held at 4 °C, but the concentration of dihydrozeatin riboside increased with ipt expression.
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Plant morphology
The overall growth habit of cor15a-ipt plants under growth chamber conditions (25 °C) was not substantially different from the wild-type chrysanthemum line (Table 3). In addition, the overall growth response of both cor15a-ipt lines and wild-type plants that were first exposed to a cold-induction treatment remained similar, indicating that the increase in ipt expression in cold-induced plants did not have a long-lasting effect on subsequent plant growth. Of the growth parameters observed, only shoot fresh weight and average lateral shoot length were affected by genotype. Shoot fresh weight for cor15a-ipt line 12 was similar to the wild type while shoot fresh weight for cor15a-ipt line 9 was lower. However, average lateral shoot length for cor15a-ipt line 12 was greater than either the wild type or cor15-ipt line 9. Shoot fresh weight and average leaf size (on the uppermost lateral branch) were both affected by cold-induction treatment, but both the cor15a-ipt lines and the wild-type plants responded in the same way to this treatment. Most significantly there was no interactive effect of genotype and environmental treatment on any of the growth responses observed, indicating that any increase in cytokinin that resulted from a cold-induction period did not persist during plant development at normal greenhouse temperatures. The average number of lateral branches on each plant, the number of secondary branches on each lateral shoot, and average internode length on the top lateral branch were all unaffected by genotype or temperature treatment.
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No differences were observed between non-induced wild type and cor15a-ipt petunia lines grown in the 25 °C growth chamber. For example, the average length of the main stem of non-induced wild-type (21±3.0 cm) and cor15a-ipt petunia plants (20.1±0.6 cm) were similar. Likewise, the average number of lateral shoots on the main stem (5.8±1.2) and (6.8±0.4), and the average internode length on the main stem (1.7±0.3 cm and 1.5±0.1 cm) were also similar for wild-type and cor15a-ipt petunia plants, respectively. Even when exposed to an initial cold treatment, growth response was similar for the wild-type and the cor15a-ipt transgenic petunia lines for four of the six parameters measured (length of the main stem, number of leaves on the main shoot, leaf area on the main shoot, and number of lateral branches on the main shoot). Compared with the wild type, shorter internodes were observed on two of the three transgenic lines tested and one transgenic line displayed a leaf area increase on the first lateral shoot. None of these anatomical features were consistent with the type of changes associated with constitutive ipt gene expression.
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Discussion |
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In the horticulture trade, billions of plugs (e.g. whole plants used for transplant) and vegetative cuttings are produced annually for sale to commercial growers. Stockpiling plugs and cuttings for later use is advantageous because it increases productivity. However, long-term storage can only be successful if the majority of the plants survive and remain vigorous. Such storage requires cold temperatures to minimize respiration and the rapid deterioration of chlorophyll that results when plants are exposed to low light and warm temperatures (Heins et al., 1995
).
The control of ipt expression, and consequently delayed leaf senescence, under specific stress conditions using the cold-inducible promoter from the cor15a gene from Arabidopsis thaliana, is demonstrated here. This promoter was selected so that gene expression would occur only after the plants were exposed to a brief but specific environmental stress. The cor15a gene is a member of the COR (cold-regulated) gene family. Cor15a encodes a 15 kDa polypeptide that is targeted to the chloroplasts. Upon transit into the organelle, the cor15a peptide is processed to a 9.4 kDa polypeptide designated as cor15am. The constitutive expression of cor15a in non-acclimated transgenic Arabidopsis plants increases the freezing tolerance of both chloroplasts frozen in situ and isolated leaf protoplasts frozen in vitro by 1–2 °C over the temperature range of –4 to –8 °C (Thomashow, 1999). Baker et al. (1994) showed that the cor15a promoter is inactive, or very weakly active, in most of the tissues and plant organs maintained under temperatures associated with active growth and, that in response to low temperature, it becomes highly activate in the shoots but not in the roots (Baker et al., 1994). Root expression of ipt is a concern with asexually propagated species because of the potential for cytokinins to impede root development. Analysis of the cis-elements within the cor15a promoter indicated that the 5' region between nucleotides –305 and +78 imparted ABA- and drought-regulated gene expression in addition to cold-regulated expression (Baker et al., 1994). Therefore, to avoid undesirable stress, which could also affect ipt gene expression, all morphological experiments were carried out under carefully controlled environmental conditions.
In these experiments, RT-PCR analysis confirmed that ipt expression was a result of cold-activation and no transcript was detected in either wild-type plants or transgenic plants that were not exposed to cold temperature conditions. These results are in accordance with the data reported by Hajela et al. (1990), who detected cor transcripts (regulated by the cor15a promoter) 1–4 h after Arabidopsis plants were exposed to cold temperatures. The amount of transcripts continued to increase for about 12 h and then remained elevated as long as the plants remained in the cold (up to 14 d in their study). However, when the plants were returned to normal growth temperatures, transcripts decreased rapidly and returned to concentrations found in the non-transgenic plants after 8 h.
These experiments showed that expression of the ipt gene in petunia resulted in an immediate increase of zeatin and dihydrozeatin type cytokinins. A 5.3-fold increase of physiologically active forms (trans-zeatin, dihydrozeatin and their ribosides), a 3.6-fold increase of storage forms (O-glucosides of trans-zeatin and dihydrozeatin and trans-zeatin nucleotide) and a 5.4-fold increase of deactivation forms (N-glucosides of trans-zeatin and dihydrozeatin) were found. The increase in the concentration of isopentenyladenine, but not of its derivatives, indicates that cytokinin metabolism, including the hydroxylation of the isoprenoid side chain, is very fast in this species. High concentrations of isopentenyladenosine under growth permissive conditions is in accordance with the results of Auer et al. (1999), who found an increase in isopentenyladenine/isopentenyladenosine in Petunia hybrida explants during shoot induction and especially in the shoot developmental phase. When considering the relatively moderate increase of endogenous cytokinins that followed cold-induced ipt expression, it is also necessary to consider that the increase in cytokinin biosynthesis probably stimulated additional cytokinin oxidase/dehydrogenase activity, in a way similar to what was detected in petunia after application of BA (Auer et al., 1999).
In chrysanthemum plants, ipt expression led to the accumulation of storage cytokinins (O-glucosides), and only after prolonged cold induction (more than 7 d), an increase in active cytokinins occurred. Short induction (3.5 d) followed by the plant transfer to growth permissive conditions (25 °C) resulted in the marked increase of all physiologically active cytokinins, accompanied by the decrease of cytokinin O-glucosides. This difference between petunia and chrysanthemum plants may be due to specific differences in the adaptation to cold temperatures. In chrysanthemum the concentration of active cytokinins seems to be tightly regulated in response to the temperature. A decrease in concentration of active cytokinin species during prolonged incubation at 25 °C would be expected if the storage temperature led to a decrease in transcript in conjunction with continued cytokinin turnover (affected by cytokinin oxidase/dehydrogenase).
In this study, an overall increase in cytokinin concentrations in cold-induced cor15a-ipt petunia and chrysanthemum plants did not reach the level reported after ipt overexpression in other systems. For example when ipt was placed under the control of a Drosophila heat-inducible promoter (hsp70) and introduced in Nicotiana plumbaginifolia, the resulting increase in cytokinin concentration ranged from 140- to 200-fold compared with non-induced leaves (Smigocki, 1991). The resulting transgenic plants were shorter, had an underdeveloped root system, and reduced leaf width. Another transgenic tobacco containing the maize hsp70-ipt gene exhibited an after-heat-treatment increase in zeatin and zeatin riboside concentrations of 52- and 23-fold, respectively (Medford et al., 1989). In these studies, a consistent, low level of expression was observed even under non-inducing conditions and plant phenotype was dramatically affected, especially at the higher cytokinin concentrations (Medford et al., 1989). When a more tightly regulated soybean heat-inducible promoter was used to regulate ipt expression, ipt transcription was not detected in plants exposed to normal temperatures and plants did not display the phenotypic characteristics associated with constant ipt expression. After heat shock, zeatin riboside concentration increased only 5-fold and the plants developed with shorted internodes, crinkled and down-folded leaves and enlarged stems. Transgenic plants also displayed delayed leaf senescence and flower bud development (Ainley et al., 1993). Still others have reported that a sharp, transient increase in cytokinins was sufficient to promote plant cell division (Redig et al., 1996; Dobrev et al., 2002), and even a temporary increase in cytokinin triggered changes in organ initiation and differentiation (Kaminek et al., 1997).
Regardless of the magnitude of changes in cytokinin concentrations observed in cor15a-ipt petunia and chrysanthemum lines, the cold-induced plants in this study displayed a dramatic increase in chlorophyll retention and a dramatic delay in senescence under warm, dark storage conditions. For example, following exposure to a 72 h activation period at 4 °C, leaves from cor15a-ipt petunia and chrysanthemum remained healthy and green even after 3 weeks of dark incubation at 25 °C. Leaves of non-transformed plants senesced under the same storage regime. Similar responses were observed for shoot tip cuttings and whole plants. In addition, actively growing cor15a-ipt lines exhibited growth and development characteristics that were similar to the wild-type petunia and chrysanthemum phenotypes. A normal phenotype was observed even when cor15a-ipt lines were initially exposed to cold activation temperatures before growing in the 25 °C environment. Thus, up-regulation of cor15a-ipt in response to cold-induction appeared to be sufficient to alter leaf senescence properties of petunia and chrysanthemum but, under light and temperature conditions associated with active growth, the presence of the cor15a-ipt gene did not elicit the type of undesirable phenomic responses associated with constitutive ipt expression.
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Acknowledgements |
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The authors thank Dr Richard Mercier (Plant Science Department, UCONN) for help in the non-radioactive detection of the ipt fragment in transgenic plants and Dr Carol Auer (Plant Science Department, UCONN) for excellent scientific discussions concerning the topic of this paper. This study was supported by a grant from Connecticut Innovation Inc.
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