Effects of Gibberellin and Several Growth Retardants on Plastid Ultrastructure in the Hepatic Marchantia polymorpha

Exogenous gibberellic acid application induced ultrastructural changes in chloroplasts of Marchantia polymorpha. Starch grains disappeared and membrane development was accompanied by an increase in granal thickness and length until dense, parallel arrays of thylakoids extended throughout the plastid. This event was associated with concomitant modification of the stroma; what was previously electron translucent became electron dense. The growth retardants HIMT, CCC and Phosfon D (4-hydroxy-5-isopropyl-2-methylphenyl trimethylammonium chloride-i -piperidine carboxylate, 2-chloroethyl-trimethylammonium chloride and 2,4-dichlorobenzyltributylphosphonium chloride, respectively) repressed the ultrastructural variation associated with gibberellin. The relevance of this response to normal morphogenesis in this hepatic is discussed. The presence and physiological activity of gibberellin in nonvascular plants has been widely observed. Radley (1961) demonstrated gibberellin biosynthesis in the brown alga Fucus vesiculosus. Exogenous gibberellin applications elicited substantial dry weight increases in Ulothrix (Conrad et al., 1959) and Gymnodinium breve exhibited gibberellin-enhanced growth (Pastor & Abbott, 1970). Jennings (1971) noted that CCC prevented gametophytic growth in Ecklonia radiata, but simultaneous GA3 application suppressed this inhibitory effect. Bud formation in protonemata of Pohlia nutans was enhanced by GA3 (Mitra & Allsopp, 1959). Gibberellin-induced development in Splachnum ampullaceum was associated with increased cell number and size (von Maltzahn & MacQuarrie, 1958). Stem elongation in Pellia epiphylla was promoted by gibberellin (Asprey et al., 1958). Endogenous gibberellin-like substances were isolated from Marchantia polymorpha by Melstrom et al. (1974). The nature and concentration of these gibberellin fractions was photoperiodically dependent, and their presence was reflected in the growth habit and coloration of the thallus. Gibberellin antagonists repressed the morphological response and lowered hormone levels. Observations of Wellburn et al. (1973) suggest that gibberellin mediated plastid development through augmented membrane growth. Earlier, Fredericq and DeGreef 'The technical advice of Russell A. Parham during the course of this study is gratefully acknowledged. 2 Department of Biology, Lawrence University, Appleton, Wisconsin 54911. 3 The Institute of Paper Chemistry, Appleton, Wisconsin 54911. This content downloaded from 157.55. 39.162 on Thu, 11 Aug 2016 05:53:52 UTC All use subject to http://about.jstor. org/terms 1976] ALBERTINE, ET AL.: MARCHANTIA PLASTID ULTRASTRUCTURE 23 (1968) had observed that 730 nm light decreased the chlorophyll content in Marchantia thalli. This response was repressed by a subsequent exposure to 660 nm light. It was later shown (DeGreef et al., 1971) that such phytochrome-induced senescence in M. polymorpha was associated with bleaching of the basal parts of the thallus caused by chloroplast degeneration (ribosome and starch loss, lipid globule appearance and vesticulation of grana and lamellae). The number of grana per choloroplast and chlorophyll content simultaneously decreased. Mache and Loiseaux (1973) observed that growth and photosynthesis in Marchantia were inhibited by light saturation in excess of 2-3 x 103 lux and that chloroplast development was altered. Vesiculation was observed; but instead of membrane degradation, enhancement appeared to occur in the grana so that grana stacks extended the length of the plastid. The established relationship between gibberellin levels and light treatment, and the observed influence of light on structure and function of Marchantia chloroplasts implied that some developmental interrelationship might be operative. This report describes the ultrastructural influence of gibberellin on Marchantia polymorpha chloroplasts. MATERIAL AND METHODS Vegetative male Marchantia thalli were kept on a 12 hr. photoperiod and cultured as previously described (Melstrom et al., 1974). Aqueous solutions of GA3 (Sigma Chemical Co.) at 10-3, 10-4 and 10-' M concentrations were sprayed onto 3-week-old vegetative thalli three times a week for each experiment. Control plants were sprayed with distilled water. The growth retardants CCC (10-2 M), HIMT (10-' M) and Phosfon D (10`" M) (West & Fall, 1972) were similarly applied alone and in conjunction with each GA3 concentration. Chemical names for the growth retardants are 4-hydroxy-5-isopropyl-2-methylphenyl trimethylammonium chloride-1-piperidine carboxylate, 2-chloroethyl-trimethylammonium chloride and 2,4-dichlorobenzyltributylphosphonium chloride, respectively. Evaluation of ultrastructural responses was made after 5 and 19 days. Mature male thallus sections 10 mm wide were excised from a region 5 mm below the apex and the lateral edges trimmed back 1 mm to remove the youngest tissue. Thallus pieces 1 mm2 were prepared from the remaining tissue for electron microscopy by prefixation in 0.1 M phosphatebuffered 2.5% glutaraldehyde (pH 7) for 3 hr. at 4 C (Millonig, 1961) and then washed in the same buffer and postfixed in 0.1 M phosphate-buffered 2% OsO for 1 hr. at 20 C (pH 7). The thallus material was dehydrated in a graded acetone/water series and embedded in Spurr Low-Viscosity Embedding Media, Modification E (E. R. Fullam, Inc.) (Dawes, 1971). Blocks were sectioned on a Porter-Blum MT-2B ultramicrotome equipped with a diamond knife. Mounted sections were stained with uranyl acetate and lead citrate (Hayat, 1972), examined with an RCA EMU-3F electron microscope at 50 kv, chloroplast morphology noted and the number of starch grains in 20 sections from each growth regulator treatment recorded. OBSERVATIONS General morphology. -Marchantia polymorpha thalli displayed a horizontal growth habit with rounded lobes. During the course of the experiment 2-3 gemma cups developed on most plants; sexual development was absent. Three distinct growth responses corresponding with 10-3, 10-4 and 10-5 M GA3 treatments were noted. Tissue continuously treated with 10-3 M GA3 was shorter than 10-4 M GA3 treated thalli. The 10-3 M concentration also made the tissue brittle. These plants exhibited a slight orthogeotropic habit. Whereas 10-3 M GA3 treatment stunted growth, 10-4 M GA3 stimulated it. When compared with untreated tissue, the thalli were longer and broader and displayed This content downloaded from 157.55. 39.162 on Thu, 11 Aug 2016 05:53:52 UTC All use subject to http://about.jstor. org/terms 24 THE BRYOLOGIST [Volume 79