Journal of Experimental Botany, Vol. 53, No. 369, pp. 621-629,
April 1, 2002
© 2002 Oxford University Press
Original Papers |
Local expression of the ipt gene in transgenic tobacco (Nicotiana tabacum L. cv. SR1) axillary buds establishes a role for cytokinins in tuberization and sink formation
1 University Pierre and Marie Curie, Laboratory CEMV, Bât. N2, 4, place Jussieu, F-75252 Paris Cedex 05, France
2 Universität Regensburg, Lehrstuhl fuer Zellbiologie und Pflanzenphysiologie, Universitaetsstrasse 31, D-93053 Regensburg, Germany
3 Universität Tübingen, ZMBP, Allgemeine Genetik, Auf der Morgenstelle 28, D-72076 Tübingen, Germany
Received 1 October 2001; Accepted 9 November 2001
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Abstract |
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The developmental characteristics of a transgenic tobacco line (BIK62) expressing the ipt cytokinin-biosynthetic gene under the control of a tagged promoter were analysed. In situ hybridization and cytokinin immunocytochemistry revealed that the ipt gene was mainly expressed in the axillary buds after the floral transition. The ipt-expressing axillary buds presented morphological alterations such as short and narrow scale-leaflets, and swollen internodes filled with starch grains, giving rise to short and tuberized lateral branches. In addition, the modification of the endogenous cytokinin balance in the axillary meristems resulted in a fast rate of leaf initiation and cytokinins accumulated mostly in the lateral zones of the reactivated axillary meristems, suggesting a role in leaf organogenesis. Cell cycle analysis revealed that the reactivated axillary meristems were characterized by predominant S+G2 nuclei. Terminal internodes displayed low levels of hexose and sucrose concomitant with starch accumulation. Extracellular invertases (EC 3.1.26) were also present in higher amounts in the tuberizing internodes compared to the axillary buds of wild-type tobacco. These results underline the role of cytokinins in cell cycle regulation and in the creation of a sink–source effect. They also provide new information about cytokinin involvement in the process of tuberization and their overproduction in axillary buds giving rise to tuberized lateral branches in a naturally non-tuberizing species.
Key words: Apical dominance, axillary bud, cytokinins, ipt gene, tuberization.
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Introduction |
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Cytokinins belong to a class of plant hormones first noted as able to promote cell division (Miller et al., 1955
). Since many data indicate that cytokinins are also active throughout plant development, controlling processes as diverse as apical dominance, root formation, leaf senescence, stomatal behaviour, and chloroplast development (Mok, 1994). More recently, cytokinins have been implicated in carbohydrate transport and metabolism and in sink–source effects (Roitsch and Ehneß, 2000). Cytokinins are also necessary at the very early stages of potato tuber formation, probably because of their role in stimulating cell division and radial cell growth, and later in stimulating starch synthase activity (Smith and Palmer, 1970). Despite extensive work to elucidate the role of cytokinins in plant development, the molecular mechanisms of their action are still unclear (Brault et al., 1997; Schmülling et al., 1999). One approach to investigate the function of cytokinins was the generation of transgenic plants overproducing cytokinins by expressing the Agrobacterium T-DNA-derived ipt gene, which encodes an isopentenyltransferase that catalyses the rate-limiting step of cytokinin biosynthesis (Akiyoshi et al., 1984; Barry et al., 1984). In order to obviate the pleiotropic effects of systemic ipt gene expression (Klee and Estelle, 1991), a number of attempts were made to express the gene conditionally or in a spatially and temporally distinct manner. In this line of research, transgenic tobacco plants expressing the ipt gene under the control of heat-regulated (Medford et al., 1989; Schmülling et al., 1989; Smart et al., 1991; Van Loven et al., 1993; Rupp et al., 1999), tetracycline-regulated (Faiss et al., 1997) or copper-regulated promoters (MacKenzie et al., 1998) were created. In addition, the possibility of inducing targeted and locally restricted cytokinin effects was demonstrated in transgenic plants that expressed the ipt gene under the control of a senescence-specific promoter. The transgenic plants showed a delay in leaf senescence in the absence of other detrimental developmental effects (Gan and Amasino, 1995). However, none of these approaches demonstrated a putative role of cytokinins in regulating sink–source relations at the whole plant level.
Tobacco transgenic lines expressing the ipt gene in specific tissues were also obtained by using a promoter tagging strategy (Hewelt et al., 1994). A promoterless ipt gene located close to the right T-DNA border was inserted randomly in the genome of tobacco plants, causing transcriptional fusions between plant promoters and the ipt gene. One particular line, named BIK62, was specifically characterized by the breaking of axillary bud dormancy. This particular phenotype was already visible in hemizygote plants, proving that it is a dominant gain-of-function phenotype. It suggested local expression of the ipt gene in the axillary buds and the transcription of the ipt gene has been demonstrated. However, the precise sites of ipt expression were not determined and significant differences in the levels of endogenous cytokinins in wild-type and BIK62 mature vegetative plants were not found (Hewelt et al., 1994). This line appeared to be suitable to study in more detail the local consequences of enhanced cytokinin production in axillary meristems. In the present paper, ipt gene expression was visualized in the axillary buds and found to be developmentally regulated. Local cytokinin overproduction, demonstrated by in situ detection techniques, was found to stimulate the cell cycling activity of the axillary meristems and the rate of leaf initiation, and to induce a range of traits usually related to the process of tuberization such as reduced longitudinal growth accompanied by stimulation of radial growth, starch deposition and scale-shaped leaves (Gregory, 1965). The tuberizing effects were accompanied by sugar mobilization. Such a phenomena have hitherto not been described for a non-tuberizing species. The effect of cytokinins on different parameters of this new developmental programme will be discussed, as well as their role in sink creation.
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Materials and methods |
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Plant material
Hemizygote transgenic tobacco (Nicotiana tabacum L. cv. SR1) line BIK62 harbouring one functional T-DNA insert has been described previously (Hewelt et al., 1994
). N. tabacum cv. SR1 was used as a control. Seeds were sown in a greenhouse, with day and night temperatures of about 25 °C and 20 °C, respectively. The 16 h photoperiod consisted of natural daylight supplemented with Mazdafluor Cool White and Durolux Truelite tubes dispensing 50 µmol m-2 s-1. Plants were subcultured 2 and 4 weeks after sowing. Growth parameters were determined from sowing to flowering.
For decapitation experiments, 10 WT vegetative and flowering plants have been sectionned below the fourth internode and examined 2 weeks later. The effect of exogenous cytokinins on the phenotype of reactivated WT axillary buds was checked on microcuttings cultivated in vitro excised from sterile micropropagated plantlets and placed on MS medium (Murashige and Skoog, 1962) containing 30 g l-1 sucrose, 0.8% agar and 1 mg l-1 BAP and adjusted to pH 5.6. The in vitro cultures were grown under a 16 h photoperiod at 25±1 °C with 70 µE m-2 s-1 irradiance.
In situ hybridization
The coding sequence of the ipt gene was cloned into a pBluescript vector (Stratagene, La Jolla, USA) which yielded plasmid p
ipt-script (A Hewelt and T Schmülling, unpublished data). This plasmid was used to generate sense and antisense RNA probes labelled with digoxigenin (Boehringer Mannheim, Mannheim, Germany) according to the instructions of the manufacturer.
For in situ hybridization, paraffin-embedded sections (7 µm) were treated as described earlier (Guivarc'h et al., 1996). Briefly, after an overnight hybridization at 55 °C, sections were extensively washed with SSC buffer then incubated with 20 mg ml-1 RNase A for 30 min. They were incubated with anti-digoxigenin antibody coupled to alkaline phosphatase (Boehringer Mannheim, diluted to 1 U ml-1). Visualization of alkaline phosphatase activity was performed by incubation (16 h) with 5-bromo-4-chloro-3-indolyl phosphate (BCIP, BioRad, Ivry-sur-Seine, France) and nitroblue tetrazolium (NBT, BioRad). Hybridization with the sense probe was used as control.
Cytokinin immunocytochemistry
Immunolocalization of cytokinin bases was carried out (Dewitte et al., 1999). Cytokinin bases were fixed in the samples using 0.5% glutaraldehyde and 3% paraformaldehyde in PBS during 2.5 h at 4 °C. 30 min of vacuum infiltration was applied at the beginning of fixation. Fixed material was then sectioned (50 µm) with a vibratome (Vibratome 1000, Technical Products International, Inc., St Louis, USA). After washing twice in PBS, the sections were incubated three times for 10 min in blocking buffer, treated for 20 min with 0.07% saponin in PBS, then incubated overnight at 4 °C with rabbit primary antibodies (anti-zeatin (Z), anti-dihydrozeatin (DHZ) or anti-isopentenyladenine (iP)) diluted in blocking buffer (1:100). Upon rinsing with PBS, the sections were incubated with the secondary antibody (alkaline phosphatase linked sheep anti-rabbit IgG, Boehringer Mannheim, diluted at 1:250). After washing three times for 10 min in PBS, the sections were transferred in TRIS buffer pH 9.6, then the immunoreactive sites were revealed using NBT and BCIP as chromogenic substrates. The sections were mounted in PBS containing 2 mM EDTA and 50% glycerol (v/v). Sections incubated without the primary antibodies were used as controls.
Nuclear DNA imaging analysis
Axillary buds excised from vegetative or flowering plants were fixed in acetic acid:ethanol (1:3, v/v), rinsed with 70% (v/v) ethanol and submitted to the Feulgen reaction (1 N HCl hydrolysis for 10 min at 60 °C, then Schiff's staining for 2 h). The stained meristems were then dissected, flattened and mounted in DePex (Gurr, BDH, Poole, UK). DNA quantification was performed with a SAMBA 2005 microspectrophotometric system (Samba Technologies, Grenoble, France), fitted with the Ploidy 4.04 software (Samba Technologies). The mean 2C reference value (±2 SD) was determined by analysis of half telophases on all types of meristems and submitted to the Kolmogorov–Smirnoff test of normality (Samba Technologies Stat 2005 software). For each condition, about 600 interphase nuclei were analysed and the relative frequency of G0/G1 and S plus G2 nuclei was calculated from histograms.
Orthophosphate determination
The leaf tissue was homogenized in ice-cold 0.2 N perchloric acid. The extract was kept on ice for 15 min, then centrifuged (13000 g, 15 min). The pellet was re-extracted twice for 15 min in perchloric acid then the supernatants were combined and analysed for total leaf inorganic phosphate (Pi) as described previously (Ames, 1966).
Carbohydrate analysis
For starch histochemical staining, samples were fixed with neutral formalin–absolute ethanol–acetic acid (3/1/1, by vol.) then paraffin-embedded and sectioned (7 µm). Sections were treated according to the periodic acid-Schiff's reaction (PAS; MacManus, 1948).
For biochemical assays, axillary buds were harvested, quickly frozen in liquid nitrogen and stored at -80 °C. Powdered frozen material (80–100 mg fresh weight) was transferred to 0.5 ml hot 80% ethanol and extracted for 20 min at 80 °C. The extract was centrifuged 10 min at 13000 g, the precipitate was re-extracted twice more, each time with 0.5 ml ethanol. The supernatant was used for soluble sugar determination and the last pellet was redissolved in distilled water for starch determination.
For soluble sugar analysis, the three supernatant fractions were pooled and evaporated under vacuum to dryness (Speedvac; Savant Instruments, New York, USA) and the residue was dissolved in 1 ml H2O. Sucrose, glucose and fructose contents were determined enzymatically by a spectrophotometric assay of NAD+ reduction as described previously (Bergmeyer and Bernt, 1974) using sugar determination kits (Boehringer Mannheim).
Starch content was determined in the ethanol insoluble fraction after incubation with 0.5 ml KOH for 20 min at 100 °C. The pH was adjusted to 6 with 1 M acetic acid and the gelatinized starch was hydrolysed with amyloglucosidase (9 IU) and determined enzymatically as glucose using sugar determination kits (Boehringer Mannheim). Data were expressed as the mean of three replicates.
SDS-PAGE and Western blotting
For cell wall protein extraction, axillary buds (100 mg) were ground in the presence of liquid nitrogen then homogenized in an homogenization buffer (Roitsch et al., 1995). Homogenates were centrifuged (14000 g, 5 min, 4 °C) and the pellets were washed three times with ice-cold water. Pellets were then resuspended in 250 µl of sample buffer (Laemmli, 1970) without ß-mercaptoethanol and centrifuged at 14000 g for 5 min. Protein samples were separated on a 12.5% SDS-PAGE minigel after incubation at 95 °C for 10 min.
Western blotting was performed as described earlier (Towbin et al., 1979). A purified extract of the CIN1 extracellular invertase (EC 3.1.26) from Chenopodium rubrum (Roitsch et al., 1995) was used as a positive control. The nitrocellulose membrane was preincubated overnight at 4 °C with 5% non-fat dried milk in TBS buffer then washed for 10 min in TBS buffer containing 0.05% Tween 20. The nitrocellulose sheet was incubated for 3 h at room temperature with the CIN1 antibody (dilution 1:1000). After washes and incubation for 1 h at room temperature with a secondary antibody (anti-rabbit IgG-AP, Boehringer Mannheim) diluted 1:2000 with PBS, immunodetection of alkaline phosphatase activity was done with BCIP and NBT according to the manufacturer's protocol (Bio-Rad).
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Results |
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Phenotypic characterization of BIK62 transgenic plants
Seeds of line BIK62 germinated at the same time as wild-type (WT) seeds, but initiated one more leaf (three instead of two) within the first 2 weeks. Later, during the vegetative phase, both WT and BIK62 showed similar phenotypes for both the aerial part and the root system (data not shown). No visible development of axillary buds was observed in both lines until flowering. After 7 weeks, both WT and BIK62 plants reached similar heights (67±15 and 75±15 cm, respectively). The first visible flower buds on WT plants were observed 9 weeks after sowing on plants provided with 17±1 leaves. The BIK62 plants flowered 1 week earlier after the initiation of the same number of leaves. This suggests that leaf number was a determinant for the onset of flowering in SR1 tobacco plants. Most of the early BIK62 floral buds became necrotic and fell off, while later appearing buds developed into longistylic flowers. Seed number was similar in WT and BIK62. Taken together, this comparison indicated a largely unaffected vegetative growth phase for plants of clone BIK62.
The most striking difference compared to WT plants concerned the behaviour of the axillary buds during the floral phase. Only one or two buds located at the axils of the more apical leaves developed into floral branches in WT plants while more basal buds remained inhibited (Fig. 1A). By contrast, all the BIK62 axillary buds initiated growth following floral transition and gave rise to short and plump lateral branches having very small and narrow leaves and short internodes (Fig. 1B, C). The axillary buds located in the upper part of the plants developed small and abnormal flower buds while basal buds remained vegetative. Several abnormalities could be observed for the flowers such as absence of reproductive organs, pale-green sepals and precocious abscission. The lateral branches of the lower buds ramified intensively giving rise to clusters of short branches that stopped growing after having reached a length of about 3 cm (Fig. 1C). All short lateral branches developed rapidly, initiating up to 40 scale-shaped leaves within about 4 weeks. In addition, these modified leaves displayed a reduced dorsiventrality with ramified and larger epidermal trichomes (not shown). Such a phenotype was never observed for the WT axillary buds reactivated upon apical decapitation of vegetative or floral plants nor for excised WT axillary buds grown in vitro in the presence of exogenous cytokinins. In both experimental situations, leaves developed by the lateral branches displayed a WT phenotype.
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Patterns of ipt gene expression in BIK62 plants
In situ hybridization (ISH) was carried out in order to determine whether the morphological modifications were correlated with a distinct pattern of ipt gene expression in axillary buds. No detectable signal was found following ISH with ipt antisense RNA probes applied to the axillary buds or to the main shoot apices during the vegetative growth phase (not shown). By contrast, lateral branches of reproductive BIK62 plants showed a hybridization signal in their apical part, including the peripheral cells of the meristem itself and the young leaf primordia, while signal was not observed in the differentiated stem and leaves (Fig. 1D
). The corresponding controls with sense probe were negative (Fig. 1E). Signal was not detected in the main shoot floral apex (not shown). Taken together, ipt expression driven by the bik62 promoter appeared to be lateral bud-specific and developmentally regulated.
In situ localization of endogenous cytokinins in WT and BIK62 axillary buds
The three cytokinin bases (iP, Z and DHZ) were detected by in situ localization and gave similar results. While signal was not detected in the control sections (Fig. 1F), a weak purple staining was observed in inhibited axillary buds of WT plants, both in the vegetative (not shown) and the flowering phase (Fig. 1G). A similar immuno-signal was observed in the inhibited BIK62 axillary buds during the vegetative phase (not shown). When the transgenic plants entered the floral phase, an increased signal was observed in the lateral buds before any bud outgrowth became visible externally (Fig. 1H). At this stage, cytokinins were detected mainly in the lateral zones of the meristem (Fig. 1H, arrows) and in the procambial strands (Fig. 1H). Later, when lateral axes were developed, a very strong signal characterized their apical meristem, the leaf primordia and the swollen subapical internodes (Fig. 1I). At the end of their development, the tuberized lateral branches contained very high amounts of polyphenols, which oxidized into dark compounds (Fig. 1J) rendering in situ localization of cytokinins impossible at this stage.
Cell cycling activity in the axillary buds
The non-developing axillary meristems located at the basal part of 7-week-old WT plants showed 95.5% of the nuclei arrested in G0/G1 (Fig. 2A). By contrast, the reactivating BIK62 axillary meristems displayed 70.5% of the nuclei in G0/G1 and 29.5% in the S and G2 phases (Fig. 2B). The percentage of S- and G2-phase nuclei was high compared to the main shoot apical meristems of vegetative tobacco plants in which only 14.8% S/G2 nuclei are present (Dewitte et al., 1999). Following floral transition, the BIK62 axillary meristems of both the apical and basal plant part expanded, reaching 140±35.7 µm and 192.5±49 µm mean width, respectively. The corresponding WT lateral meristems were only 82.6±21 and 107.1±35.3 µm wide, respectively. Occasionally, the expanded meristems underwent a process of dichotomization leading to two meristems of normal size (not shown).
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Tuberization traits of the developing BIK62 lateral branches
Structural analysis of the BIK62 short lateral branches revealed a range of particular traits that fitted very closely with the events that classically define tuberization. Microscopic examination of both longitudinal and transversal sections of 3–4-week-old short lateral branches showed a swelling of the internodes located below the apical meristem. While the diameter of the WT internodes usually increased from the tip to the base, the BIK62 lateral axes displayed a larger diameter in their apical internodes (1920±200 µm) than in their basal part (1280±125 µm). The young internodes were provided with enlarged and flat pith cells suggesting that the swelling was due to radial extension of the pith cells while their longitudinal growth was inhibited (Fig. 1I). Such swelling affected only the terminal internodes of the lateral branches as shown by the smaller stem diameter of the basal internodes (Fig. 1J). In addition, the tuberized internodes contained many large starch grains in the pith (Fig. 1K) and cortical cells; smaller starch grains were present in the meristem itself. By contrast, no starch accumulation occurred in the internodes of WT axillary buds reactivated upon decapitation (Fig. 1L). In addition, very few xylem elements were formed along the whole axis of the BIK62 lateral branches and no secondary xylem differentiated while the vascular cylinder contained abnormally extended phloem tissue. Continuous vascular cambium did not develop, even at the bases of relatively normally developed branches. Following 4–5 weeks of vegetative growth, apical leaves of the BIK62 tuberized branches dried and became brown.
Altered carbohydrate composition in BIK62 axillary buds
After 6 weeks of vegetative growth, carbohydrate determination in non-growing axillary buds showed a 5-fold higher starch content and a 2-fold lower sucrose content in the BIK62 buds compared to the WT, while the concentrations of the reducing sugars, glucose and fructose, remained identical (Fig. 3A, B). Therefore, despite the absence of significant phenotypic differences between WT and BIK62 plants during the vegetative phase, metabolic changes had already occurred, suggesting an early but undetectable expression of the ipt gene. This finding was further confirmed by the analysis of sugar and orthophosphate (Pi) content in fully expanded leaves of 6-weeks-old plants (Table 1). BIK62 leaves contained a 58–61% higher content in both sucrose and starch. Concomitantly, a significant lower amount in Pi was detected in the BIK62 leaves (Table 1).
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After 10 weeks, when both WT and BIK62 transgenic plants had entered the flowering phase, sugar determination was performed in the axillary buds located in upper, middle and lower part of the plants (Fig. 3A
, B). All the non-growing WT axillary buds were characterized by low starch content (around 3 mg eq glucose g-1 FW) and very high glucose (around 11.4 mg eq glucose g-1 FW) and sucrose (around 4.5 mg eq glucose g-1 FW) contents, irrespective of their position along the stem. By contrast, the growing BIK62 axillary buds contained very high levels of starch. 44%, 34% and 21% of the assimilate were partitioned into starch in the upper, middle and lower buds, respectively, compared to the WT. Glucose and sucrose were both present at lower levels in BIK62 buds (around 2 mg eq glucose g-1 FW) than in the WT. The data indicated for BIK62 an inverse relationship between sucrose and starch content versus glucose and fructose content, depending on the stem position of the axillary buds.
Increased extracellular invertase levels in BIK62 axillary buds
Sink capacity in developing tubers is potentialy limited by sucrose metabolism and/or starch synthesis. Sucrose synthase (Zrenner et al., 1995) and extracellular invertase (Tang et al., 1999; Roitsch et al., 2000) are both involved in sucrose partitioning. As mRNAs for extracellular invertase were also induced by cytokinins, the level of extracellular invertase as a potential indicator of the sink capacity was also examined. Western blotting for extracellular invertase performed on axillary buds showed an increased level in the BIK62 line which could be detected at the end of the vegetative phase (Fig. 4). Indeed, at 6 weeks, despite the fact that the BIK62 axillary buds were not yet developed, they exhibited a high level of extracellular invertase while only a low signal was detected in WT. After 12 weeks, when the BIK62 axillary buds were tuberizing, they all exhibited a stronger invertase signal than their WT counterpart. In WT plants, the invertase signal was slightly higher in the axillary buds of the upper part of the plants. In BIK62 plants, a very strong signal characterized all the axillary buds, whatever their position on the stem.
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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This work described novel developmental effects caused by a local cytokinin overproduction in axillary buds of transgenic tobacco plants through promoter tagging with the ipt gene. The expression of the bik62 promoter was shown to be chiefly axillary bud-specific and activated upon floral transition, causing a highly localized enhancement of cytokinin production in the axillary buds. These axillary buds developed into short and tuberized axes with numerous scale-shaped leaflets, revealing the plasticity of developmental potential in tobacco. This particular phenotype of the lateral branches was never observed in transgenic plants expressing the ipt gene in the whole plant (Klee and Estelle, 1991
; Schmülling et al., 1989; Faiss et al., 1999; Rupp et al., 1999) nor in WT decapitated plants or in microcuttings grown in the presence of exogenous cytokinins. It appears to be the consequence of the local cytokinin overproduction that becomes significant upon the floral transition in the BIK62 axillary buds. Some metabolic modifications were found during the vegetative phase confirming the previous finding of ipt gene expression in early developmental stages (Hewelt et al., 1994). However, this expression was probably too weak either to be detected by ISH or to produce a phenotype. Given that WT tobacco axillary buds are usually stimulated during the floral phase (Smigocki, 1995), the new phenotype observed in the BIK62 plants could result from both the cytokinin overproduction and from a developmentally regulated change in cytokinin sensitivity.
Tuberization is a complex developmental process that was mainly studied in potato. Early physiological studies (Smith and Palmer, 1970) and more recent approaches with ipt transgenic potato (Ooms and Lenton, 1985; Gális et al., 1995) have involved cytokinins in this process. In tobacco, the present paper demonstrates that localized ipt gene expression caused developmental changes in tobacco axillary buds similar to those occurring during tuberization in a species for which tuberization is not a natural and inherent property.
The ability to tuberize seems to be a characteristic of species, rather than of a family or a genus (Gregory, 1965). For tobacco species, one might predict that it is not competent to develop a tuberization programme. However, the tuberizing effects of cytokinin overproduction in axillary buds suggests that a single dominant factor is able to trigger the tuberization process. Apparently, locally enhanced cytokinin production is a necessary and sufficient requirement to stimulate tuberization. Cytokinins are perhaps not the only determinants of in vivo tuberization, but the local effects of their overproduction in the lateral buds mimics the whole range of parameters that are usually used to define tuberization. The locally and temporally specific cytokinin overproduction appears to give priority to these buds as a sink for available assimilates in the plant, while growth of the main axis is maintained.
Tuberization involves well-defined and concerted changes in sugar metabolism, leading to a concomitant decrease in metabolites (Davis, 1984). These data on sugar accumulation support the hypothesis that cytokinins are involved in the regulation of competition for assimilates (Kuiper, 1993) and in the creation of sinks by regulating the expression of genes implicated in the assimilate partitioning and source–sink regulation such as invertases and hexoses transporters genes (Ehneß and Roitsch, 1997; Roitsch and Ehneß, 2000; Roitsch et al., 2000). In the BIK62 plants, local modification of the endogenous cytokinin level results in a new distribution of the assimilates in favour of the cytokinin-enriched axillary buds. This sink effect is evidenced by both the decrease in hexose and sucrose levels concomitant with starch accumulation and by a higher level of extracellular invertase. In addition, the different gradients of glucose, sucrose and fructose in buds from the upper to the lower parts of BIK62 plants suggest the existence of regulatory signals at the whole plant level. These data support the hypothesis of links between cytokinins and the establishment of a sink effect involving extracellular invertases. Modifications of the patterns of sugar accumulation in the cytokinin-producing branches indicate that carbon fluxes can be redirected through genetic engineering with tissue-specific promoters fused to the ipt gene.
Other novel cytokinin effects were observed, such as an enlargement of the axillary meristems, sometimes accompanied by a process of dichotomization. This alternative to fasciation in enlarged meristems has already been described for in vitro-grown tobacco buds in the presence of high cytokinin levels (Brossard, 1976) and in the Arabidopsis mutant mgoun (Laufs et al., 1998). Enhanced cytokinin levels also increased the percentage of S+G2 nuclei in the growing axillary meristem. This could be due to cytokinins controlling the cell cycle G1 to S transition through cyclins D, as reported recently (Riou-Khamlichi et al., 1999). Finally, the question of whether all the new traits observed in the BIK62 axillary branches result directly from cytokinin overproduction is still open as recent works have suggested reciprocal links between cytokinins and homeotic genes (Frugis et al., 1999; Rupp et al., 1999).
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Acknowledgments |
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The authors are grateful to Dr A Hewelt for the gift of plasmid p
ipt-script and Professor H Van Onckelen for providing the anti-cytokinin antibodies. They also thank Catherine Scott-Taggart for proofreading. |
Notes |
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4 To whom correspondence should be addressed. Fax: +3310144274582. E-mail: anne.guivarch{at}snv.jussieu.fr
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