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Journal of Experimental Botany, Vol. 53, No. 375, pp. 1747-1751, August 1, 2002
© 2002 Oxford University Press

The role of gibberellins in embryo axis development

Received 4 March 2002; Accepted 29 March 2002

Dirk B. Hays1, Edward C. Yeung and Richard P. Pharis

Plant Physiology Research Group, Department of Biological Sciences, 2500 University Drive NW, University of Calgary, Calgary, Alberta, Canada T2N IN4

Abbreviations: DAC, days after culture (for microspore-derived embryos); DAP, days after pollination; MDE(s), microspore-derived embryo(s); SNK, Student–Newman–Keuls; ZEs, zygotic embryo(s).


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 References
 
The role of gibberellins (GAs) during early embryo development was examined using microspore-derived embryos (MDEs) of Brassica napus. At the globular stage of development, 10 d after initial culture (DAC) when endogenous GA1 levels are increasing rapidly, a triazole, uniconazole, was used at 1, 33 and 100 µM to inhibit GA biosynthesis. Within this dose range there was no apparent effect of the inhibitor on embryo growth through to the early torpedo stage. However, by 25 DAC uniconazole-treated MDEs showed significantly reduced (50%) axis elongation. Addition of GA1 at 33 µM on 14 DAC to embryos pretreated with 1 µM uniconazole on 10 DAC prevented this reduction in axis length, giving axis elongation equivalent to untreated MDEs. Application of GA1 alone, however, did not significantly increase axis elongation. The reduced axis growth seen with uniconazole treatment was due to reduced cell elongation, but not cell number, and the co-applied GA1 thus prevented the uniconazole-induced reduction in cell length. The elongating axis of MDEs may thus be a useful tool for examining the role of GAs in cell elongation.

Key words: Key words: Brassica napus, embryo development, gibberellins, microspore-derived embryos (MDEs), uniconazole.


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 References
 
To date, no study has definitively established a role for GAs during zygotic embryogenesis. This is at least partly due to the inaccessibility of the embryo during embryogenesis. However, by examining the developmental pattern of the suspensor, an organ very rich in GAs, during embryo development several workers have obtained at least a partial answer for the physiological role(s) that GAs may play during embryo development for Phaseolus (Alpi et al., 1975), Tropaeolum (Picciarelli et al., 1984) and Cytisus (Picciarelli et al., 1991). In crucifers the suspensor reaches maximal cell number by the globular stage of development and begins to senesce by the end of the torpedo to early cotyledonary stage (Yeung and Meinke, 1993; Mansfield and Briarty, 1991). These latter two stages also represent the end of axis elongation in crucifers (Tykarska, 1976, 1979). While the above studies have demonstrated the importance of suspensors as a source of GAs, no study has actually demonstrated that GAs play a physiological role during embryo development. Therefore, the use of microspore-derived embryos (MDEs) of Brassica napus have been used as a model for assessing the putative role(s) of GAs in embryo development, and especially in embryo axis elongation.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 References
 
Plant growth conditions
Brassica napus cv. Topas (seed obtained from Dr Keith Downey, Agriculture Canada, Saskatoon, Saskatchewan) plants were grown at 25/16 °C day/night temperature with a 16 h photoperiod (400 mmol m–2 s–1) for 5 weeks and then transferred to low temperature (12/7 °C day/night) until whole flower buds were harvested.

Chemicals and growth regulators
Sucrose for the MDE medium (Lichter, 1982) was purchased from BDH (Toronto, Ontario, Canada) and other chemicals were analytical grade. The early stage GA biosynthesis inhibitor, uniconazole, was a gift from Sumitomo Chemical Co., Osaka, Japan.

Microspore-derived embryo (MDE) culture
Microspore-derived embryos were cultured according to Hays et al. (1996, 1999). Briefly, microspores were isolated from flower buds (2–3 mm long) of cold-treated plants (see above) by grinding the buds in NLN 13% sucrose medium (Lichter, 1982) at pH 6.0, then washing and pelleting the microspores (220 g for 5 min) in the same medium. The microspores were then resuspended in 40% Percoll containing 13% sucrose (BDH), overlaid with NLN medium and spun at 220 g, for 10 min. Microspores at the Percoll–media interface were collected, washed, pelleted, and resuspended in fresh NLN medium. Ten ml of the resupended microspores were then plated in Petri dishes at a density of 3x104 cells ml–1 and incubated in the dark for 4 d at 30 °C, after which they were transferred to 25 °C and subcultured for another 4 d in the dark, but with fresh NLN medium.

For the uniconazole treatment MDEs were collected at 10 d after initial culture (DAC) and recultured for varying periods at 1, 33 and 100 µM concentrations of uniconazole. In order to assess the ability of a GA to restore growth of uniconazole-treated MDEs, 1 or 33 µM GA1 was added at 14 DAC to cultures that had been grown on 1 µM uniconazole since 10 DAC.

Histological preparations
Control and uniconazole-treated MDEs were collected at 25 DAC. All tissues were prepared according to Yeung and Law (1987), for example, tissues were fixed in 50 mM phosphate buffer, pH 6.8 containing 2.5% glutaraldehyde and 1.6% paraformaldehyde for 24 h at 4 °C, then dehydrated in methyl cellosolve for 24 h. Methyl cellosolve was then replaced with absolute ethanol with two changes over 48 h.

Infiltration of LKB Historesin was carried out at 4 °C with daily changes at progressively higher concentrations of Historesin (3:1, 1:1, 1:3, pure Historesin). The pure Historesin solution was changed once more and left at 4 °C for 3 d. This tissue was then embedded in 10 ml of Historesin infiltration solution, to which was added 0.6 ml of hardener and 0.4 ml PEG 200 (Yeung and Law, 1987). The mixture was allowed to polymerize for 5 h in plastic moulding trays with microtome mounting chucks. The polymerized tissue was then sectioned at 3 µm with a Reichert 2040 Autocut microtome using a Ralph-type glass knife. The sections were stained with Periodic acid–Schiff’s (PAS) reaction for total carbohydrates and counter-stained with toluidine blue O (TBO) for general histological organization or amido black 10B for proteins (Yeung, 1984). The preparations were examined and photographed with a Leitz Aristoplan light microscope. The images were recorded using Kodak Technical Pan film. Cell expansion in the MDE cotyledons was measured using a Zeiss light microscope with a built-in calibrated eyepiece micrometer. Sections were used which were cut in a longitudinal median with respect to the embryo axis. Cell elongation measurements were made from the point of cotyledon attachment to the embryo axis, down to the root meristem. A minimum of 10 median sections were used for each measurement representing 10 individuals for each treatment.


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 References
 
One way to examine the effect of GAs during embryogenesis is to use an inhibitor of GA biosynthesis. Uniconazole, a triazole which is known to inhibit the oxidative conversion of ent-kaurene to ent-kaurenoic acid, was chosen (Izuma et al., 1985; Fletcher and Gilley, 2000). In both the MDEs and zygotic embryos, GA1 (the endogenous, growth-active GA of the Brassica embryo) begins to increase significantly by 10 DAC, which is the globular stage of embryo development (Hays et al., 2001). Ten DAC was thus chosen as the initial time point to begin examining the role of GAs in embryo development. When uniconazole was applied to MDE cultures at 10 DAC across a range of concentrations (1–100 µM), it had no obvious effect on embryo growth rate up to the early torpedo stage. By 25 DAC, however, uniconazole-treated MDEs showed a 50% reduction in axis elongation, relative to untreated (Control) MDEs (Fig. 1a, b).



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  		Fig. 1. (A) Photograph of MDEs with or without treatment with a range of concentrations of the GA biosynthesis inhibitor, uniconazole. Embryos are 25 DAC; all embryos were treated with 1–100 µM uniconazole at 10 DAC. (B) Effect of 1–100 µM uniconazole concentrations on the elongation of the MDE axis. Embryos were treated at 10 DAC and measured at 25 DAC. Values represent the mean of three independent experiments, with 50 embryos being measured in each replicate. Error is ±SE. Letters in each bar indicate significance groupings (P <0.05) via the SNK test.

 
The ability of applied GA1 to reverse the effect on uniconazole treatment on axis elongation was then examined. For non-uniconazole-treated ‘control’ MDEs, applied GA1 gave no significant increase in axis elongation (Fig. 2a). However, addition of 33 µM GA1 at 14 DAC to MDE cultures which had previously been treated (at 10 DAC) with 1 µM uniconazole yielded axis elongation which was statistically equivalent to the control embryos (Fig. 2b). The inhibition of axis elongation by treatment with uniconizole is accompanied by reduced cell elongation (Figs 3a, b, 4). Further, the GA1+uniconazole-treated embryos had cell lengths comparable to true control MDEs (Figs 3, 4). These effects strongly suggest that axis elongation of the Brassica embryo requires GA1.



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  		Fig. 2. (A) Effects of 1–33 µM GA1 on the elongation of the MDE axis. Embryos were treated at 10 DAC and measured at 25 DAC. Values represent the mean of three independent experiments; 50 embryos were measured in each experiment for each treatment. Error bar is ±SE. Letters above each bar indicate that GA1 had no significant effect on axis elongation (P <0.05) via the SNK test. (B) Inhibition in axis elongation by uniconazole treatment is reversible. Embryos were measured at 25 DAC. All embryos were treated with 1 µM uniconazole at 10 DAC with GA1 being added to uniconazole-treated embryos at 14 DAC. Values represent the mean of three independent experiments where 50 embryos were measured in each experiment for each treatment. Error is ±SE. Letters in each bar indicate significance groupings (P <0.05) via the SNK test.

 


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  		Fig. 3. Histological comparisons of control versus uniconazole- and GA1-treated MDEs. Embryos were treated with uniconazole at 10 DAC and harvested for serial sectioning at 25 DAC. Axis sections are longitudinal median sections with respect to the embryo axis. Sections are (A) control, (B) 1 µM uniconazole, and (C) 1 µM uniconizole plus 33 µM GA1 (GA1 being applied at 14 DAC). Micrographs are from the middle portion of the axis with respect to root and shoot apical meristems. Sections were stained with PAS for carbohydrates and amido black for proteins (Yeung and Law, 1987; Yeung, 1984). The bar equals 20 µm.

 


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  		Fig. 4. Cell elongation in the axis of MDEs. This illustration shows that inhibition of cell elongation by uniconazole treatment is reversible. Measurements of cell elongation were made along the inner cortex at 25 DAC. All embryos were treated with 1 mM uniconazole at 10 DAC. GA1 being added to uniconazole-treated embryos at 14 DAC. Values represent the mean of three independent experiments where 50 embryos were measured in each experiment for each treatment. Error is ±SE. Letters in each bar indicate significance groupings (P < 0.05) via the SNK test.

 
Through the use of cell counts it was found that the effect of uniconazole in reducing axis elongation is primarily due to decreased cell elongation, not decreased cell division. The mean number of cells per average axis length was 171±12 SE in the uniconazole-treated MDEs, whereas control MDEs had 166±10 SE cells. Applied GA1 at 33 µM had no apparent effect on cell number (174±11 SE cells per average axis length). Finally, MDEs where the uniconazole (1 µM) inhibitory effect on axis elongation had been prevented by co-application of GA1 (Fig. 2b) showed comparable cell numbers (169±16 SE cells per average axis length). Statistical analysis by the SNK test (SPSS software package, SPSS Inc., Chicago, Illinois, USA) showed that cell numbers for all MDE treatments did not differ significantly from cell numbers for control or GA1-treated MDEs.


   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
References
 
It has been shown that by reducing endogenous GA1 levels through the use of an early stage inhibitor of GA biosynthesis, uniconazole, embryo axis elongation can be significantly reduced. More specifically, based on cell length measurements, uniconazole reduced cell elongation within the axis by 50% (Fig. 4), without reducing cell division. Cell elongation has been shown to be a key component in GA-regulated stem elongation (Cosgrove and Sovonick-Dunford, 1989; Yang et al., 1996; Cowling and Harberd, 1999). This inhibitory effect of uniconazole on axis cell elongation could be prevented by co-application of GA1 to uniconazole-treated MDEs (see above and Fig. 2b), with no significant change in cell numbers.

Another triazole, paclobutrazol, was found to reduce both cell numbers and length in stems of safflower plants (Potter et al., 1993). By contrast, the reduction in length for leaves of wheat treated with paclobutrazol was brought about by reduced cell elongation, not cell number (Tonkinson et al., 1995). It appears, then, that there can be differential responses to the application of triazole inhibitors, depending perhaps upon species or organ.

Treatment of the MDEs at 10 DAC with uniconazole had no measurable effect on cotyledon expansion, which occurs at 20–25 DAP, or on cotyledon cell number (Hays et al., 2000).

An earlier study that examined GA levels in the suspensor (Alpi et al., 1975; Picciarelli et al., 1984, 1991), together with the knowledge that the attached suspensor is critical for optimal growth of isolated zygotic embryos (Corsi, 1972; Monnier, 1984; Yeung and Sussex, 1979) is also supportive of a role for endogenous GAs in embryo growth. In fact, culturing embryos at the heart stage in the presence of low concentrations of GA3, can replace the need for the suspensor (Yeung and Sussex, 1979). Interestingly, at the cotyledonary stage, when axis elongation is nearing completion, leaving the suspensor attached had little effect on embryo growth (Yeung and Sussex, 1979). Thus, the requirement for optimal endogenous GA levels in axis elongation of the zygotic embryo likely occurs between the torpedo and early cotyledenary stages.

Further evidence for a causal role for GAs in embryo axis elongation also comes from physiological comparisons of B. napus zygotic and MDE development where a major morphological difference exists between the embryo systems. In MDEs, the axis is quite elongated, relative to the lateral expansion that occurs within the cotyledon. Also, the zygotic embryos of B. napus, has no detectable GAs in the surrounding endosperm (Hays et al., 2001), while MDE cultures have a high level of growth-active GAs in the growth medium (Hays et al., 2001). Thus, the axis of the MDE may continue to grow (via cell elongation not cell division) beyond the comparable stage seen for zygotic embryos, as a consequence of the high GAs in the medium.

Uniconizole blocks the conversion of ent-kaurene to ent-kaurenoic acid via an inhibition of ent-kaurene oxidase. Copalyl diphosphate synthase (CPS) resides upstream and is considered the first committed step in the synthesis of gibberellins. A CPS gene designated GA1 has been cloned from Arabidopsis using the ga1-3 dwarf mutants with impaired seed and embryo development (Silverstone et al., 1997). The GUS reporter gene fused to the GA1 promoter expressed highest in growing tissues, such as root tips, developing flowers, seeds, and the embryo axis beginning at the heart stage to mid- to late- cotyledonary stage (Silverstone et al., 1997). These results, along with results from the present study, indicate that gibberellins are indeed synthesized in, and required for, normal cell elongation in the embryo axis.

The mechanism by which GAs promote cell elongation is still poorly understood (Cosgrove and Sovonick-Dunford, 1989). The MDE system, where embryo axis cells are dependent upon GA1 for their elongation, may thus represent a useful model system for studying the mechanism of GA-regulated cell elongation.


   Acknowledgements
 
This research was supported by operating grants from the Natural Sciences and Engineering Research Council of Canada to RP Pharis and EC Yeung.


   References
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 Materials and methods
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 Discussion
 References
 
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Cosgrove DJ, Sovonick-Dunford SA. 1989. Mechanism of gibberellin-dependent stem elongation in peas. Plant Physiology 89, 184–191.[Abstract/Free Full Text]

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