Effects of gibberellin and a gibberellin biosynthesis inhibitor on spore germination in the liverwort Fossombronia crassifolia Spruce

Erin K. Shanle*, Barbara Crandall-Stotler, Aldwin AnterolaSouthern Illinois University, Carbondale, IL, 62901

ABSTRACT - Gibberellins (GAs) are important phytohormones that are involved in stem elongation, seed germination and flower induction in seed plants, and formation of antheridia and spore germination in ferns. Although ubiquitous in vascular plants, their occurrence and potential function in bryophytes has been little studied. In mosses, a recent study showed that spore germination rates are reduced by AMO-1618, a compound that inhibits the production of ent-kaurene, a common precursor to GAs. The effect is less pronounced when ent-kaurene is also present in the growth media. Exogenous GA, however, did not promote spore germination or reduce the inhibitory affect of AMO-1618. Our experiments have now tested the effects of exogenous GA and AMO-1618 on spore germination and development in the liverwort Fossombronia crassifolia. In contrast to mosses, in this liverwort neither compound inhibits spore germination. Instead, AMO-1618 retards the growth of leafy shoots from the protonemal phase, and GA interferes with early patterns of cell division and inhibits shoot formation. These findings suggest that further study into the potential role of GAs in the growth and development of liverworts is warranted.

INTRODUCTION - Gibberellins (GAs), first isolated from the fungus Gibberella fujikuroi, have been identified in bacteria, fungi, and numerous vascular plants (MacMillan 2001). In flowering plants, GAs stimulate cell elongation and seed germination as well as regulate floral development (Pimenta Lange & Lange 2006). In ferns, they promote spore germination under dark conditions and antheridia formation (Weinberg & Voeller 1969, Yamane 1991). Previous studies by Anterola et al. (unpublished data) have shown that in the moss Physcomitrella patens spore germination rates are reduced with the application of AMO-1618, a compound that inhibits the synthesis of the ent-kaurene, an intermediate in the GA synthesis pathway. Application of exogenous GA did not affect rates of spore germination and did not reduce the inhibitory effect of AMO-1618. The influence of GA and AMO-1618 on spore germination in liverworts has never been tested. Thus, the objectives of this study were to:1. Determine whether exogenous GA or AMO-1618 affects spore germination in

the liverwort Fossombronia crassifolia Spruce. 2. Observe the effects of GA and AMO-1618 on sporeling morphology. 3. Compare the effects of GA and AMO-1618 observed in F. crassifolia with

observed effects in mosses and ferns in order to better understand the role of GAs in early lineages of land plants.

PROCEDURES - Spore germination studies:1. Nutrient culture media was prepared, following the procedure of Hatcher

(1965). For the experimental treatments, 0.1 mM GA3 or 1 mM AMO-1618 were filter-sterilized and added to the media prior to plate pouring. Six regimes were tested - control, GA, and AMO-1618 under full spectrum light and control, GA, and AMO-1618 under dark conditions (Fig. 2).

2. Six capsules were independently surface-sterilized in a laminar flow hood, using standard procedures.

3. The spores of each capsule were released into 1 mL of sterile H2O; each of 6 treatment plates was inoculated with 100 µL of the spore suspension. This was repeated for all six capsules, generating 6 replicates for each treatment. Following inoculation, plates were sealed with parafilm and dark treatment plates were wrapped in aluminum foil. All plates were randomly distributed on the same shelf of a Percival environmental chamber, 15 cm below the lights. The chamber was maintained at a 12 hr photoperiod, with a 15°C day/12°C night temperature regime.

4. After one week, the plates were examined using a light microscope to determine the number of spores on each plate. A dense area of spores was selected and monitored throughout the experiment. Every week, the spores were observed for germination. After 21 days, the proportion of germinated spores was calculated and compared among the treatments (Fig. 3).

SEM studies of sporeling morphology:After 3 months of growth, scanning electron microscopy images were taken of each treatment using a Hitachi S570 SEM. Live plants were fixed in a 2% glutaraldehyde/2% paraformaldehyde solution, buffered with 0.1 M sodium cacodylate, pH 7.2 @ 4° C for 24 hrs, post-fixed for 3 hrs at room temperature in 2% aqueous OsO4, dehydrated through a graded ethanol series, and critical point dried using CO2 as the transition fluid.

SPECIMEN INFORMATION - Fossombronia crassifolia Spruce: MEXICO, Veracruz. Hills south of Xico, nr La Molienda restaurant, 4290’, August 2008, Renzaglia s.n. (Voucher ABSH). Sporophytes that were present at the time of collection matured in an environmental chamber at SIUC; SEM micrographs were used to confirm identity (Fig. 1).

Figure 1. Fossombronia crassifo-lia Spruce; a) population with both mature and developing capsules; b) SEM of spore, distal view; c) SEM of spore, proximal view. During germination, the spore wall breaks on the proximal face.

RESULTS - Germination occurred in both control and treatment plates exposed to light, but no germination occurred in any of the dark treatment plates. There was substantial variation in the proportion of spores that germinated among the replicates of any treatment (Table 1). An ANOVA showed, however, that there was no statistical difference in germination among the treatments, with P value = 0.9258 (Table 1). After 3 months, the foil was removed from the dark treatment plates and within 3 weeks of being placed in the light, these spores also germinated. Though neither GA nor AMO-1618 affects spore germination in F. crassifolia, post-germination development is modified by the presence of these compounds (Fig. 4). After three months of growth, the plants on the control plates had developed normal shoots and rhizoids, with substantial branching and numerous cycles of leaves. The GA and AMO-1618 treatments appeared to retard development; the germinated spores remained in the sporeling stage much longer in both treatments and gametophore formation appeared to be inhibited or delayed. Greater growth inhibition was observed in the GA treatment. SEM images were taken of each light treatment in order to further identify the developmental differences among the treatments (Figure 5). The control plants showed normal, organized formation of leafy shoots and rhizoids from a defined, globose protonema. Plants grown on media containing AMO-1618 also showed formation of leafy shoots from an organized protonema, but the shoots were small and juvenile, suggestive of a developmental delay. The plants grown on media containing GA show the most significant developmental modification. From the SEM images, it appears that normal patterns of cell division in the protonemal phase are disrupted by GA and the germinated spores do not develop the apical cell system required to produce leafy shoots. These randomized cell masses ultimately turn brown and die.

Figure 2. Diagram of the experimental design.

Figure 3. Comparison of ungerminated and germinated spores; a) dark control; b) light control.

Treatment Mean Range VarianceP-Value0.9258

0.1 mM GA3, Light 0.1146 0.0479-0.3382 0.01221 mM AMO-1618, Light 0.0911 0.0385-0.2791 0.0088

Control, Light 0.1022 0.0211-0.3136 0.0112All Dark Treatments 0 0 0

Table 1. Comparison of spore germination data, 21 days after inoculation; values respresent proportions.

Figure 4. Comparison of plant growth, 3 months after inoculation; a) control; b) AMO-1618 treatment; c) GA treatment.

Figure 5. Morphological effects; a & b) control; c) AMO-1618 treatment; d) GA treatment. Normal protonema at *.

a b

a cb

a cb

c d

a b

CONCLUSIONS - Liverworts display a different response to exogenous GA3 and AMO-1618 as compared to mosses and ferns. In the latter group, GAs promote dark germination, but in F. crassifolia neither treatment eliminated the requirement of light for germination. The dramatic effect of GAs on protonemal development and bud formation suggests that they may play a role in cell division and differentiation in these earliest land plants, perhaps in conjunction with other hormones like cytokinins. Future studies of GA signalling pathways should include liverworts to determine whether the molecular basis of this response is an evolutionary precursor to the vascular plant pathway.

REFERENCES - Hatcher R. The Bryologist, 1965, 68: 227-231.MacMillan J. J Plant Growth Reg, 2001, 20(4): 387-

442. Pimenta Lange M & Lange T. Plant Biology, 2006,

8: 281-290. Weinberg E & Voeller B. PNAS, 1969, 64: 835-842. Yamane H. Gibberellins. Springer-Verlag, New

York: 378-388.

ACKNOWLEDGEMENTS - Financial support for this project

was provided by National Science Foundation grant, EF-0531750. We thank K. Renzaglia (SIUC) for providing the collection of F. crassifolia.

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