Organogenic responses of Pinus pinea cotyledons to hormonal treatments: BA metabolism and cytokinin content

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1 Tree Physiology 25, Heron Publishing Victoria, Canada Organogenic responses of Pinus pinea cotyledons to hormonal treatments: BA metabolism and cytokinin content P. MONCALEÁN, 1 P. ALONSO, 2 M. L. CENTENO, 3 M. CORTIZO, 2 A. RODRÍGUEZ, 2 B. FERNÁNDEZ 2 and R. J. ORDÁS 2,4 1 Instituto Vasco de Investigación y Desarrollo Agrario, Centro Arkaute, Apdo. 46, Vitoria, Pais Vasco, Spain 2 Unidad de Fisiología Vegetal, Departamento de Biología de Organismos y Sistemas, Universidad de Oviedo, C/Catedrático Rodrigo Uría, s/n Oviedo, Asturias, Spain 3 Departamento de Biología Vegetal, Universidad de León, Facultad de Ciencias Biológicas y Ambientales, Campus de Vegazana, León, Spain 4 Corresponding author (rordas@uniovi.es) Received December 19, 2003; accepted April 2, 2004; published online November 1, 2004 Summary Isolated cotyledons from mature Pinus pinea L. embryos were cultured in vitro in a factorial combination of 4.4, 10 and 44.4 µm N 6 -benzyladenine (BA) for 2, 4, 8, 16 and 35 days to optimize shoot regeneration. Incubation of explants in 44.4 µm BA for 4 days, in place of the standard incubation in 4.4 µm BA for 35 days, reduced the entire culture period to 4 weeks. Shortening the culture period had no significant effect on the caulogenic response or the number of buds formed per cotyledon. To establish the relationship between key moments in the caulogenic process induced by 4.4 µm BA and the endogenous concentrations of the active forms of BA and other isoprenoidtype cytokinins (CKs), we examined uptake, metabolism and amount of BA, as well as the amounts of zeatin, dihydrozeatin and their ribosides in P. pinea cotyledons after 1, 2, 6, 12 and 24 h, and 2, 4, 8, 16 and 35 days of exposure to 8-[ 14 C]BA. Uptake and release of BA were associated with water movement between explants and the medium during the first 8 days of culture. The interconvertible forms of BA were the main metabolites formed in the tissues. Inactivation of BA as a result of conjugation or oxidation was insignificant. The endogenous concentration of BA + N 6 -benzyladenosine was 20-fold higher than the exogenously applied BA during the competence acquisition phase (Days 0 3). The concentration of isoprenoid-type CKs also increased 16-fold and then decreased during this time. Induction of shoot buds (Days 4 8) was characterized by a second peak of BA uptake by explants that triggered the synthesis of N 6 -benzyladenosine-5 -monophosphate and by the maintenance of isoprenoid-type CKs. Reestablishment of CK homeostasis marked the shift from the induction phase to the shoot development phase in this organogenic process (Days 8 12). Keywords: BA uptake, organogenesis, shoot regeneration. Introduction Commercial use of in vitro techniques to clone and mass propagate superior genotypes of forest tree species is dependent on a reliable culture system that supports a high frequency of plant production. Pinus spp. propagation systems are based on the induction of shoot buds in cotyledonary explants dissected from embryos and cultured in the presence of a cytokinin (CK), usually N 6 -benzyladenine (BA) (Diamantoglou et al. 1990, García-Férriz et al. 1994, Capuana and Giannini 1995, González et al. 1998, Humara and Ordás 1999, Humara et al. 1999). Bud formation is dependent on the BA concentration in the medium and the exposure time of the explant to BA. Age and tissue differentiation of pine explants are other determining factors, as shown by the inverse relationship between germination time of the embryos and the shoot-forming ability of their excised cotyledons (Martínez-Pulido et al. 1990, Saravitz et al. 1991, López et al. 1996, Valdés et al. 2001). Despite these limiting factors, the culture system used for the production of stone pine (Pinus pinea L.) shoots is characterized by a high multiplication potential. Nevertheless, various aspects of the system could be improved, among which a reduction in culture time would be particularly beneficial. The caulogenesis model established for pine cotyledons is a good system for studying the control of in vitro shoot organogenesis in conifers (Thompson and Thorpe 1997, Valdés et al. 2001). The P. pinea model provides several advantages, including the uniformity and low degree of differentiation of the cells in the cotyledonary tissue, the requirement for BA as the only hormone in the shoot-induction medium, and the synchronous fashion in which cotyledonary explants respond in vitro. We used this model along with the genetic and molecular information now being obtained about the mechanisms underlying shoot organogenesis in Arabidopsis thaliana (L.) Heynh. plants and hormone-independent mutants (Sugiyama 1999, Frank et al. 2000, Banno et al. 2001, Cary et al. 2001, Che et al. 2002, Howell et al. 2003) to study the hormonal control of caulogenesis. In a previous study on the hormonal control of caulogenesis in P. pinea, we found that cotyledons taken from 0-day-old embryos (C0) had concentrations of indole-3-acetic acid and several CKs three- or fourfold higher than those of older and

2 2 MONCALEÁN ET AL. more differentiated cotyledons, and showed that this difference was related to the responsiveness of the cotyledons to BA (Valdés et al. 2001). However, the induction of the caulogenic process is controlled by dynamic biochemical pathways involving both exo- and endogenous plant growth regulators, and including uptake, distribution and metabolism of BA (Auer et al. 1992, 1999, Moncaleán et al. 1999, Feito et al. 2001, Centeno et al. 2003). Therefore, to gain further insight into the hormonal control of caulogenesis, the endogenous concentrations of active molecules, mainly CKs, within the cotyledons must be characterized throughout shoot organogenesis. Our first objective was to improve the production of P. pinea shoots by reducing the culture time of cotyledons in the presence of BA. We tested the response of the most reactive cotyledonary explants (C0) to several BA concentrations applied for different incubation periods. Our second objective was to gain insight into the reaction of C0 cotyledons to externally applied BA, and identify the putative roles of this and other CKs in the caulogenic process in conifer explants. We determined the endogenous amounts of BA and some isoprenoid-type CKs (zeatin (Z), dihydrozeatin (DHZ), zeatin riboside ([9R]Z) and dihydrozeatin riboside ([9R]DHZ) in cotyledonary explants, mainly during the phases of cell competence acquisition and shoot-bud induction. We analyzed absorption, metabolism and amounts of BA in the explants, as well as changes in the amounts of the other CKs after varying periods of culture in the presence of 4.4 µm BA. Materials and methods Plant material One-year-old mature seeds from open-pollinated stone pine (Pinus pinea L.) trees growing in a natural stand (Meseta Norte region, Spain) were provided by the Servicio de Material Genético del Ministerio de Medio Ambiente (Spain). After removal of the seed coat, megagametophytes were surfacesterilized by immersion in 7.5% (v/v) H 2 O 2 for 45 min, followed by three rinses in sterile double-distilled water. The surface-sterilized seeds were imbibed on moistened sterile paper for 48 h at 4 C. Bud induction and shoot elongation Cotyledons were excised from the embryos of the imbibed seeds and placed horizontally on 20 ml of caulogenic induction medium (1/2LPB), consisting of half-strength Le Poivre medium (1/2LP), as modified by Aitken-Christie et al. (1985), supplemented with 0.8% (w/v) Roko agar (Roko S.A., Oleiros, Spain), 3% (w/v) sucrose and 4.4, 10 or 44.4 µm BA. Before autoclaving, the ph of all media was adjusted to 5.8. Ten explants were placed in each culture vessel. To determine the minimum induction period, cotyledons were cultured on the caulogenic induction media for 2, 4, 8, 16 or 35 days. Thereafter, the cotyledons were transferred to a hormone-free medium (1/2LPC), consisting of 1/2LP supplemented with 0.5% (w/v) activated charcoal (Sigma, Madrid, Spain), 0.8% (w/v) Roko agar and 3% (w/v) sucrose. Explants were subcultured monthly onto fresh 1/2LPC medium. All plant cultures were maintained in a growth chamber at 25 ± 4 C with a 16-h photoperiod at a photon flux of 80 ± 5 µmol m 2 s 1 provided by cool white fluorescent tubes (TLD 58 W/33, Phillips, Suresnes, France). The percentage of bud-forming cotyledons and the number of shoots per explant were recorded after two subcultures on 1/2LPC (60 days). A completely randomized experimental design was applied with two subsamples (20 cotyledons) per incubation time and BA concentration. Experiments were repeated three times. Data are presented as means ± standard error (SE). Statistical analysis of qualitative data (% cotyledons forming buds) was carried out with the χ 2 test. Quantitative data (number of buds per cotyledon) were analyzed by nonparametric tests, the Kruskall-Wallis test for n independent group analyses, and the Mann-Whitney test for two independent group analyses. Differences were considered significant at the 5% level. All statistical tests were performed with SPSS statistical software (SPSS, Chicago, IL). Extraction and measurement of BA uptake To analyze BA uptake and metabolism, P. pinea cotyledons were placed in caulogenic induction medium (1/2LPB) containing a final BA concentration of 4.4 µm (1 mg l 1 ) made of 16.6 kbq of 8-[ 14 C]BA (specific activity = 2.0 GBq mmol 1, Amersham Ibérica, Madrid, Spain) with unlabeled BA. Explants cultured in the presence of labeled BA were harvested after short (1, 2, 6, 16 and 24 h) and long (2, 4, 8, 16 and 35 days) periods of culture. At least 20 cotyledons from each incubation period with 8-[ 14 C] BA were washed with distilled water for a few seconds to remove medium residues, dried with filter paper, and their fresh mass (FW) determined. Cotyledons were then frozen in liquid N 2, powdered, lyophilized and reweighed to determine their dry mass (DW) and to calculate water content per cotyledon. Experiments were repeated three times. For the three replicates of each sample, 100 mg of lyophilized tissue was extracted twice with 25 and 15 ml of 80% (v/v) methanol containing 10 mg l 1 butylated hydroxytoluene, for 14 and 7 h, respectively, by repeated inversion at 4 C in darkness. Three 1-ml aliquots were taken from the combined extracts, reduced to dryness and re-suspended in 0.5 ml of distilled water and 1.5 ml of Pico-Aqua (Packard, Frankfurt, Germany) scintillation fluid. Radioactivity was measured with a liquid scintillation spectrometer (Packard 2500 TR). Total BA uptake was determined following correction for extraction volume and sample mass. The remaining methanolic extracts were evaporated under vacuum (30 C) and the resulting aqueous solutions were brought to a final volume of 5 ml with distilled water and cleared by centrifugation (10,000 g for 15 min at 4 C). The supernatants were collected and analyzed for BA metaboliites and isoprenoid-type CKs. Analyses of BA metabolites and isoprenoid-type CKs Extracts containing labeled BA metabolites and CKs were adjusted to ph 3 with acetic acid and percolated through a 10-ml TREE PHYSIOLOGY VOLUME 25, 2005

3 CYTOKININS AND MORPHOGENESIS IN P. PINEA COTYLEDONS 3 column of monoammonium ionic-form cellulose phosphate (Whatman P11, Brentford, U.K.) equilibrated with acidified water (ph 3 with acetic acid). The CK nucleotides (acidic fraction) were then eluted with 50 ml of acidified water (ph 3). Subsequently, the CK bases and nucleosides were eluted with 50 ml of 2 M ammonium hydroxide (basic fraction). Radioactivity was measured in aliquots of the acidic and basic fractions as previously described. Solvent was evaporated, and the fractions were resuspended in 5 ml of 0.6% acetic acid (v/v) and 5 ml of distilled water, respectively. Further purification was achieved by using Sep-Pak C 18 cartridges (Waters, Milford, MA) (Feito et al. 1994). Separation of the isoprenoid-type CKs (DHZ, [9R]DHZ, Z, [9R]Z) and BA and its metabolites (adenine (Ade), adenosine (Ado), BA (authentic standards supplied by Sigma), N 6 -benzyladenosine ([9R]BA), N 6 -benzyladenine-3-glucoside ([3G]BA), N 6 -benzyladenine-7-glucoside ([7G]BA) and N 6 -benzyladenine-9-glucoside ([9G]BA) (authentic standards supplied by Apex Organics, Devon, U.K.)) was accomplished by high performance liquid chromatography (HPLC) in a Waters 600 liquid chromatograph equipped with a diode array detector (Waters 996). The column used was a Kromasil C 18 ( mm; 5 µm) and the flow rate was 1.5 ml min 1. The mobile phase was acetonitrile: triethylamine acetate buffer (TEAA, 40 mm, ph 7) in a linear gradient from 5 to 20% acetonitrile (v/v) over 20 min. To verify CK retention times, kinetin (2 µg) was added to each sample as an internal standard. The eluted CKs were monitored at 265 and 275 nm, fractions were collected at 1-min intervals and reduced to dryness by speed-vac concentration. The separation of N 6 -benzyladenosine-5 -monophosphate ([9R-5 P]BA) (Apex Organics) in the acidic fraction was accomplished with the same column. The mobile phase was methanol:aqueous buffer (0.2 M acetic acid adjusted to ph 3.5 with triethylamine). A linear solvent gradient from 5 to 45% (v/v) methanol over 45 min and a flow rate of 1 ml min 1 were used. Each basic and acidic fraction from each extract was injected on the HPLC in duplicate. Isoprenoid-type CKs, BA and its metabolites were putatively identified by co-elution with authentic standards and by their UV absorption spectra. 128 GBq mmol 1, synthesized in our laboratory) was added to the initial methanolic extracts. Recoveries ranged from 70 to 90%. Results Determination of minimum induction period with BA The BA concentration in the medium and the induction period affected the percentage of cotyledons forming buds (Figure 1A). The minimum period that explants had to be in contact with exogenously applied BA to form shoot buds was 2 days with 44.4 µm BA, 4 days with 10 µm BA and 8 days with 4.4 µm BA. The minimum induction period in which 100% of cotyledons formed buds was 2 days when they were cultured in 44.4 µm BA, 8 days in 10 µm BA or 16 days in 4.4 µm BA. The highest mean number of buds per cotyledon (Figure 1B) was obtained for cotyledons cultured 35 days in the presence of 4.4 µm (57.5 ± 21.4), followed by 35 or 16 days in 44.4 µm BA. A substantial number of buds per cotyledon (17.13 ± 2.98) was also obtained by incubation in 44.4 µm BA for 4 days. Quantification of BA metabolites and isoprenoid-type CKs Measurements of the radioactivity associated with BA and its metabolites were made in the corresponding dry HPLC fractions as described for BA uptake. Quantification was based on the percentage of the label corresponding to each radioactive HPLC fraction, the total label found in each sample, and the specific activity of the labeled compound supplied. Quantitative determination of isoprenoid-type CKs was accomplished by enzyme-linked immunosorbent assay (ELISA). The dried HPLC fractions were resuspended in Tris-saline buffer (TSB, 25 mm, ph 7.5) and quantified with polyclonal rabbit antibodies raised in our laboratory (Fernández et al. 1995). We used anti-[9r]z to measure [9R]Z and Z, and anti-[9r]dhz to measure [9R]DHZ and DHZ. Assays were performed in triplicate with Immunolon M129 Micro ELISA plates (Nunc, Slangerup, Denmark) based on the modification of the ELISA method described by Centeno et al. (1996). To calculate isoprenoid-type CK losses, 0.1 kb 3 H t-zeatin riboside ( 3 H-ZR) (specific activity Figure 1. Caulogenic response (%) (A) and mean number of buds per cotyledon (± SE) (B) in Pinus pinea cotyledons cultured in vitro in half-strength LP medium with 4.4, 10 and 44.4 µm N 6 -benzyladenine (BA) for 2, 4, 8, 16 and 35 days and then transferred to half-strength LPC medium. Data were taken after 60 days of in vitro culture. Significant differences (α = 0.05) between BA treatments are indicated by different letters in each group of bars. Significant differences (α = 0.05) between incubation periods are indicated by different numbers. TREE PHYSIOLOGY ONLINE at

4 4 MONCALEÁN ET AL. Growth and water content of cotyledons Cotyledons showed changes in water content and dry mass during culture in the presence of 4.4 µm BA for 35 days (Table 1). During the first 2 h of culture, the water content of the cotyledons increased by 21% as a result of water uptake from the medium. Cotyledon water content varied only slightly in the following 14 h of culture and then decreased by 12% between 16 h and Day 1, implying release of water from the explants. The water content increased again by 29% between Days 1 and 8 and remained constant thereafter. Despite slight fluctuations between 0 to 16 h, the dry mass of the cotyledonary explants remained almost constant until Day 4 of culture (Table 1). On Day 4, the cotyledons started to grow slowly, and the growth rate increased after Day 16. Cotyledonary dry mass increased fourfold by the end of the experiment. BA absorption The kinetics of BA uptake in P. pinea cotyledons cultured for 35 days on 1/2 LPB (4.4 µm BA) containing 8-[ 14 C] BA are shown in Figure 2. The BA was taken up quickly by explants during the first 16 h of culture. Uptake rate was 37 nmol h 1 1 g DW in the first 2 h and 11.3 nmol h 1 1 g DW in the next 14 h. From 16 h to Day 2, there was a loss of radioactivity in the tissues, which suggests that the cotyledons released BA or some BA metabolites, or both, to the culture medium (Figures 2A and 2B). Uptake of BA increased again, at a constant rate of 0.5 nmol h 1 g 1 DW, from Days 2 to 8. After that, total radioactivity in cotyledons remained constant for the following 8 days and then increased for the following 35 days. Despite this third phase of BA uptake, the endogenous concentration of BA equivalents first decreased, and then remained constant thereafter as a result of a dilution of the absorbed radioactivity as the cotyledons grew. BA metabolism The applied 8-[ 14 C] BA was metabolized by P. pinea cotyledons. We detected five peaks of radioactivity after HPLC of the basic fraction of the extracts, corresponding to Ade + Ado, [3G]BA, [7G]BA, [9G]BA and BA + [9R]BA based on their Table 1. Dry mass (means ± SE) and water content of Pinus pinea cotyledons cultured in vitro with 4.4 µm N 6 -benzyladenine (BA) for different induction periods. Significant differences (α = 0.05) between treatments are indicated by different letters. Incubation Dry mass per Water period cotyledon (mg) content (%) 0 h 1.86 ± 0.04 a a 1 h 1.33 ± 0.08 b c 2 h 1.50 ± 0.07 a d 6 h 1.31 ± 0.09 b c 16 h 1.75 ± 0.34 a c 1 day 1.77 ± 0.15 a b 2 days 1.82 ± 0.15 a c 4 days 1.80 ± 0.06 a d 8 days 2.12 ± 0.03 c e 16 days 3.15 ± 0.22 d e 35 days 7.36 ± 0.00 e e retention times and UV-absorption spectra. The radioactivity found in the acidic fraction was associated with a single peak that was putatively identified as [9R-5 P]BA (Figure 3). The BA nucleotide and free BA + [9R]BA were the major metabolites present in the samples, with the percentage of radioactivity associated with the other BA metabolites being less than 12% at all times assayed. The metabolites [7G]BA and [3G]BA were only found in explants after 4 and 16 days of culture, respectively, and in small amounts, whereas [9G]BA appeared during the first hours of culture in trace amounts. The proportion of radioactivity corresponding to Ade + Ado remained unchanged or increased transiently, but never represented more than 10% of the total BA taken up (Figure 3). Metabolism of BA in the cotyledons showed three phases. During the first phase, from 0 to 16 h (Figure 3A), most of the radioactivity corresponded to free BA + [9R]BA, and the percentage of radioactivity associated with [9R-5 P]BA did not exceed 13%, although this nucleotide was synthesized by the explants during the first hour of culture. After 16 h of culture, BA metabolism changed, coinciding with the beginning of the loss in water and radioactivity in the cotyledons. The second phase, 16 h to Day 8, was characterized by a marked increase Figure 2. Amount of N 6 -benzyladenine (BA) taken up by Pinus pinea cotyledons cultured in vitro in half-strength LPB during the first hours (0 24 h) (A) and during the entire 35 days of culture (B). Values are means ± SE. TREE PHYSIOLOGY VOLUME 25, 2005

5 CYTOKININS AND MORPHOGENESIS IN P. PINEA COTYLEDONS 5 Figure 3. Dynamics of the N 6 -benzyladenine (BA) metabolites present in Pinus pinea cotyledons cultured in vitro in half-strength LPC medium during the first 24 h (A) and during the entire 35 days of culture (B). Values, which are means of three experiments, are expressed as percentages of the total radioactivity present in the basic and acidic fractions. Abbreviations: Ade + Ado = adenine + adenosine; [3G]BA = N 6 -benzyladenine-3-glucoside; [7G]BA = N 6 -benzyladenine-7-glucoside; [9G]BA = N 6 -benzyladenine-9-glucoside; BA + [9R]BA = N 6 - benzyladenine + N 6 -benzyladenosine; and [9R-5 P]BA = N 6 -benzyladenosine-5 -monophosphate. in [9R-5 P]BA, which accounted for 78% of the radioactivity in explants by Day 8, and by a dramatic decrease in BA + [9R]BA (from 80% of radioactivity at 16 h to 6% by Day 8) (Figures 3A and 3B). Thus, the BA initially absorbed by the cotyledons remained in the tissues as a free base or was converted to [9R]BA, whereas the BA absorbed between Days 2 and 8 was mainly transformed to [9R-5 P]BA. The BA metabolism changed again after Day 8 of culture when the cotyledons stopped taking up BA from the medium. Between Days 8 and 16, the percentage of BA + [9R]BA in explants was enhanced as a result of the hydrolysis of [9R-5 P]BA, which decreased to 52% of the radioactivity by Day 16. Thereafter, the radioactivity associated with each fraction remained almost constant. Uptake and metabolism of BA changed the endogenous concentrations and contents of [9R-5 P]BA and BA + [9R]BA in P. pinea explants during the 35-day culture period, as shown in Table 2. The initial phase of BA uptake into cotyledons was accompanied by a large increase in the endogenous concentration of of BA + [9R]BA, which reached a maximum of 88 µm in the explants at 16 h. At this time, the endogenous concentration was 20-fold higher than the BA concentration present in the medium (4.4 µm). Between 24 h and Day 2 of culture, the endogenous concentration of BA + [9R]BA declined sharply to 25.3 µm, and then declined at a lower rate during the following 6 days, reaching 3.6 µm by Day 8. The endogenous concentration of [9R-5 P]BA increased to 27 µm during the first days of culture. It then declined until the second phase of BA uptake into the tissues, which caused the endogenous concentration of [9R-5 P]BA to increase to 27 µm, which was maintained until Day 8, in contrast to the pattern observed for BA + [9R]BA. Although the endogenous concentrations of BA + [9R]BA and [9R-5 P]BA increased during the last period of culture as a consequence of the third phase of BA uptake, the concentrations were reduced by half because of the dilution resulting from growth of the cotyledonary tissue. Isoprenoid-type CKs Table 3 shows the concentrations of isoprenoid-type CKs in P. pinea cotyledons during the 35 days of culture in 1/2LPB Table 2. Metabolite BA + [9R]BA and [9R-5 P]BA concentrations and contents in cotyledonary tissues of Pinus pinea cultured in vitro with 4.4 µm BA for different incubation periods. Values are means ± SE. Within a column, significant differences (α = 0.05) are indicated by different letters. Abbreviations: BA = N 6 -benzyladenine; [9R]BA = N 6 -benzyladenosine; and [9R-5 P] = N 6 -benzyladenosine-5 -monophosphate. Incubation period Concentration (µm) Content (pmol cotyledon 1 ) BA + [9R]BA [9R-5 P]BA BA + [9R]BA [9R-5 P]BA 0 h 0.00 ± 0.00 f 0.00 ± 0.00 e 0.00 ± 0.00 e 0.00 ± 0.00 f 1 h ± 3.60 cd 2.90 ± 0.70 de ± 3.12 d 6.64 ± 1.46 e 2 h ± 3.20 d 3.58 ± 0.76 d ± 4.24 d ± 2.22 e 6 h ± 2.60 b 4.17 ± 0.68 d ± 8.26 c 9.10 ± 0.67 e 16 h ± 10.5 a ± 1.80 c ± a ± 4.05 d 24 h ± 8.70 a ± 3.21 ab ± b ± 7.81 d 2 days ± 3.80 c ± 2.10 c ± 6.32 c ± 5.64 d 4 days ± 2.10 d ± 2.01 a ± 7.96 c ± c 8 days 3.56 ± 0.45 f ± 2.00 c ± 2.20 d ± b 16 days 7.24 ± 1.30 e ± 1.70 c ± b ± b 35 days 2.89 ± 0.03 f ± 1.67 c ± b ± a TREE PHYSIOLOGY ONLINE at

6 6 MONCALEÁN ET AL. Table 3. Endogenous concentrations of cytokinins in Pinus pinea cotyledons cultured in vitro with 4.4 µm N 6 -benzyladenine (BA) during different incubation periods. Values are means ± SE. Within a column, different letters indicate significant differences (α = 0.05). Abbreviations: Z = zeatin; [9R]Z = zeatin riboside; DHZ = dihydrozeatin; and [9R]DHZ = dihydrozeatin riboside. Incubation period Cytokinin (pmol g 1 DW ) Z [9R]Z DHZ [9R]DHZ Total 0 h ± 7.77 f ± b ± 2.25 c ± 3.10 de ± cd 1 h ± b ± 8.51 b ± 2.30 c ± 3.85 b ± b 2 h ± a ± a ± 2.17 a ± a ± a 6 h ± d ± 8.18 bc ± 1.86 c ± 3.02 d ± d 16 h ± c ± 6.62 c ± 5.81 b ± 5.39 cd ± cd 1 day ± d ± 2.03 c ± 1.60 c ± 0.77 f ± d 2 days ± de ± 1.07 bc ± 2.10 c ± 2.37 d ± d 4 days ± ef ± 4.39 c ± 8.88 a ± 2.79 e ± cd 8 days ± ef ± 9.88 c 9.97 ± 2.17 c ± 2.21 d ± d 16 days ± f ± 7.43 b ± 1.82 c ± 2.79 e ± d 35 days ± g ± 1.17 d ± 3.80 c ± 5.84 c ± d (4.4 µm BA). Zeatin was present in higher concentrations than [9R]Z, DHZ and [9R]DHZ at all times that the samples were assayed. Endogenous concentrations of the four CKs increased between 0 and 2 h of culture, with the concentrations of Z and [9R]DHZ increasing 26- and 18-fold, respectively. Concentrations of [9R]Z, DHZ and [9R]DHZ returned to initial values during the following 4 h. Although the Z concentration decreased dramatically between 2 and 6 h of culture, it remained twice as high as at 0 h. Subsequently, there were slight decreases in concentrations of the CKs, with the exception of a second peak in Z concentration at 16 h, which was lower than the first peak, and a second peak in DHZ concentration at Day 4. Concentrations of isoprenoid-type CKs measured at 8 days and thereafter were similar to, or even lower than, those present in the initial explants. Discussion Clonal micropropagation of P. pinea can be achieved by in vitro culture of excised cotyledons in the presence of BA, which facilitates the induction of adventitious shoot buds that develop microshoots that can be rooted. We have improved the standard culture system (López et al.1996, González et al. 1998, Valdés et al. 2001) by reducing the period for bud induction from 5 weeks to 4 days. This was possible by increasing the BA concentration in the medium tenfold (Figure 1A). Although the number of buds formed by each cotyledon was lower in explants incubated for 4 days in 44.4 µm BA than in explants cultured for 35 days in 4.4 µm (Figure 1B), the first treatment is preferred because it reduces the total culture time by at least 4 weeks. Moreover, long exposures of cotyledons to high BA concentrations can lead to callus formation and development of hyperhydric shoots, as observed in Pinus ayacahuite (C.A.) Ehrenb. (Saborio et al. 1997). Other studies have shown that a short pulse of BA at a high concentration is more effective than a low BA concentration over an extended period for inducing adventitious shoot buds on cotyledons of Pinus spp. (Jang and Tainter 1991), Pseudotsuga menziesii (Mirb.) Franco (Goldfarb et al. 1991) and Picea sitchensis (Bong.) Carrière (Drake et al. 1997). Our findings support those of Christianson and Warnick (1983, 1985) that shoot organogenesis in tissue culture is a multistep process. During the first 8 days of culture in the presence of 4.4 µm BA, the P. pinea cotyledons acquired competence to respond to organogenic signals and became committed to producing shoots (Figure 1A). Although these two phases occurred under the same hormonal conditions, they can be dissected. It is clear that competence acquisition, defined as the dedifferentiation and proliferation of some cells, took place during the first 3 days of culture, because cell divisions were observed in cotyledons at this time (histological data not shown), but no buds developed if explants were transferred after 4 days to a medium without BA. Shoot commitment or induction, in which the fate of competent cells is specified for the formation of shoot buds, occurred midway between Days 4 and 8 of culture. A further experiment showed that 12 days of incubation in 4.4 µm BA was enough to achieve induction in 100% of the cotyledons, indicating that this second phase (shoot commitment) was completed by Day 12 of culture. During the third phase, which occurs after Day 12, the determined meristems develop into complete shoots independently of the exogenous supply of phytohormones. In an attempt to relate the endogenous concentrations of active CKs with the key periods of shoot organogenesis, we analyzed the uptake and metabolism of C 14 -labeled BA in P. pinea cotyledons, and determined the endogenous concentrations of Z, DHZ, [9R]Z and [9R]DHZ during culture in the induction medium. The study revealed bidirectional transport of BA or some BA metabolite, or both, between the medium and the explants, and also showed that BA was absorbed in three phases (Figure 2). We assumed that the rapid movement of BA during the first 2 days of culture occurred by diffusion and mass flow, because the uptake and subsequent release of radioactivity were accompanied by influx into and subsequent efflux from the cotyledons (Table 1). Between Days 2 and 8, a second phase of BA uptake occurred by mass flow, but this could not have happened by diffusion, because the concentration gradient of BA was unfavorable for its diffusion into the TREE PHYSIOLOGY VOLUME 25, 2005

7 CYTOKININS AND MORPHOGENESIS IN P. PINEA COTYLEDONS 7 cotyledons (Table 2). Because CK bases and ribosides migrate easily across the plasma membrane, whereas nucleotides and glucosides do not (Laloue and Pethe 1982, Kamínek et al. 1997, Mok and Mok 2001, Schulz et al. 2001), CK transport in P. pinea explants probably affected BA and [9R]BA. If so, BA + [9R]BA taken up between Days 2 and 8 was entrapped by cells after metabolic conversion to the polar [9R-5 P]BA. Cotyledonary growth should increase the absorption surface during the last 19 days of culture. This growth is likely to have caused the third phase of BA uptake in explants, which occurred after a period in which no BA was taken up by the explants. In cultured cotyledons, BA metabolism can be related to the low degree of differentiation of embryo tissues (Hoth et al. 2003). Explants mainly transformed the BA absorbed to nucleoside ([9R]BA) and nucleotide ([9R-5 P]BA) conjugates that can be easily interconverted among themselves and with BA (Chen 1997, Strnad 1997, Mok and Mok 2001, Haberer and Kieber 2002). Neither the formation of irreversible products of BA conjugation, such as 7- and 9- N-glucosides, nor the degradation of BA to adenylic compounds (Ade and Ado) was significant (Figure 3). Therefore, the strong organogenic response shown by cultured cotyledons of stone pine was largely determined by the low rate of BA inactivation through N-glucosylation or oxidation, allowing the BA taken up to remain in an active form or as BA-releasing molecules. The formation and hydrolysis of [9R-5 P]BA played a key role in BA homeostasis and mobilization within the P. pinea explants, as exemplified by the following observations. (1) The accumulation of [9R-5 P]BA was triggered after 16 h of culture to remove the excess BA + [9R]BA resulting from the first phase of BA absorption. (2) The amount of BA + [9R]BA continued to be reduced through nucleotide synthesis until Day 8, even though an increase was expected in response to the second phase of BA uptake. (3) The [9R-5 P]BA was hydrolyzed midway between Days 8 and 16, providing the BA + [9R]BA needed to establish and maintain an equilibrium of BA + [9R]BA inside and outside the explants. Thus, cellular efflux and BA nucleotide conversions appear to be the two major processes regulating CK activity derived from exogenous BA in P. pinea explants. The importance of BA nucleotides in regulating CK activity has been noted previously in several cultured angiosperm tissues capable of caulogesis (Auer et al. 1992, Centeno et al. 2003). Similarly, Zhang et al. (2003), working with relatively undifferentiated bud tissues of Pinus radiata (D. Don), also postulated that the turnover rate of phosphorylated CKs regulates CK activity during in vivo organogenic processes. Uptake and metabolism of BA by P. pinea explants led to two sharp peaks in the endogenous concentration of BA or BA-releasing molecules, or both, at h of culture and at Day 8 (Table 2). Day 8 coincides with the minimum period of exposure to 4.4 µm BA required for bud induction. The first peak of CK activity corresponded to BA + [9R]BA, and preceded the onset of proliferation by 48 h, indicating that these compounds probably promoted competence acquisition by cells and induced them to divide. Isoprenoid CKs from endogenous sources (mainly Z, which increased in concentration before the onset of cell proliferation (Table 3)) may also contribute to this function. These suppositions are supported by the proved participation of CK bases in cell division (Riou- Khamlichi et al. 1999, Laureys et al. 1998, Stals and Inzé 2001). Moreover, accumulation of free bases of isoprenoid or aromatic CKs has been correlated previously with the start of cell proliferation in other culture systems including partially synchronized tobacco cells (Reding et al. 1996) and kiwifruit tissues and callus (Centeno et al. 2003). The period of caulogenetic induction in P. pinea explants (from Day 4 to 8 of culture) was characterized by enhanced metabolic control of BA uptake as indicated by its conversion to [9R-5 P]BA. Thesedataimply that [9R-5 P]BAwastheactive form of BA for shoot commitment. A similar suggestion was made by Auer et al. (1992), who detected BA nucleotides, but not free BA, during shoot induction in cultured tissues of two Petunia hybrida Hort. Vilm.-Andr. lines. Nevertheless, Yamada et al. (2001) demonstrated that the CRE1/AHK4/ WOL gene, the CK receptor identified in CK-independent mutants of Arabidopsis (Inoue et al. 2001), recognized CK bases but not CK nucleotides, suggesting that they are not active molecules. Another explanation for the role of [9R-5 P]BA during bud induction might be that competent cells need to accumulate lypophobic and easily hydrolyzable BA metabolite as a stable pool from which the active form of BA is derived in low amounts. Our results support this suggestion. The shift from the induction phase to the shoot development phase in P. pinea explants, which occurred around Day 8, coincided with the end of the fluctuations in water content and with the reestablishment of CK homeostasis including both isoprenoid and aromatic CKs. Thus, our results agree with those of Kaminek et al. (1997) who proposed that rapid and transient changes in CK concentration initiate developmental processes such as organ formation, whereas hormonal homeostasis is required for further development of the initiated events. Acknowledgments We thank Dr. Jaime Menéndez-Humara for reviewing the English of this manuscript. This research was partly funded by the Ministerio de Ciencia y Tecnología (BOS and AGL ). References Aitken-Christie, J., A.P. Singh, K.J. Horgan and T. Thorpe Explant developmental state and shoot formation in Pinus radiata cotyledons. Bot. Gaz. 146: Auer, C.A., J.D. Cohen, M. Laloue and T.J. Cooke Comparison of benzyladenine metabolism in two Petunia hybrida lines differing in shoot organogenesis. Plant Physiol. 98: Auer, C.A., V. Motyka, A. Brezinová and M. Kamínek Endogenous cytokinin accumulation and cytokinin oxidase activity during shoot organogenesis of Petunia hybrida. Physiol. Plant. 105: Banno, H., Y. Ikeda, Q.-W. Niu and N.-H. 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