Somatic hybridisation with aphototropic mutants of the moss Ceratodon purpureus: Genome size, phytochrome photoreversibility, tip-cell phototropism and chlorophyll regulation

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© 1998 by Gustav Fischer Verlag, Jena

Somatic Hybridisation with Aphototropic Mutants of the Moss Ceratodon purpureus: Genome Size, Phytochrome Photoreversibility, Tip-cell Phototropism and Chlorophyll Regulation T. 1 2

LAMPARTER

1

*, G. BrUcker!' H. Eschl, J. Hughes!, A. Meiste.-2, and E.

HARTMANN

1

Institut fur Pflanzenphysiologie, Freie Universitat Berlin, Konigin Luise Str. 12-16, D-14195 Berlin, Germany Institut fur Pflanzengenetik und Kuiturpflanzenforschung (IPK), Corrensstr. 3, D-06466 Gatersleben, Germany

Received June 3, 1997 . Accepted October 25, 1997

Summary

This paper describes somatic fusion of protoplasts in the moss Ceratodon purpureus, specifically of the phototropic mutants ptr103 and ptr116. Phototropism of Ceratodon filaments is phytochrome mediated. The ptr116 mutant is thought to carry a lesion in the biosynthetic pathway of the phytochrome chromophore; this mutant grows negatively gravitropically in continuous unilateral red light. The ptr103 mutation is thought to effect phytochrome signal transduction; the phenotype of this mutant is clearly distinguish-able from wildtype and ptr116 because filaments grow in random directions in continuous unilateral red. Following protoplasting and PEG-mediated protoplast fusion with ptr103 and ptr 116, lines with a wildtype-like phenotype were identified, propagated and tested for genome size and phytochrome physiology. The 1C genome size of Ceratodon is estimated to lie between 240 and 270 Mbp, based on comparisons with Arabidopsis thaliana (genome size 100 Mbp). The genome size of one line that arose from a fusion experiment, ptr103(:t )ptr116, was shown by flow cytometry to be ca. twice that of the wildtype. With respect to phytochrome spectral activity, phototropism and regulation of chlorophyll synthesis, ptr103( + )ptr 116 was similar to the wildtype. These data are consistent with ptr103( +)ptr 116 being a bona .fide somatic fusion product in which each mutant parent complements a recessive genetic lesion of the other.

Key words: Ceratodon purpureus, moss, aphototropic mutant, genome size, flow cytometry, phototropism, phytochrome. Abbreviations: MA =absorbance change between the Pfr and the Pr form of phytochrome; CV =coefficient of variance; DAP! =4',6-diamidino-2-phenylindole; n.d. =not determined; SE =standard error of the mean. Introduction

Growth direction of protonemal tip cells of the leafy moss

Ceratodon purpureus is controlled by both light and gravity

(Hartmann et al., 1983; Schwuchow et al., 1990). The phototropic response of these cells is mediated by the red light

* Correspondence.

J Piant.J?hys;oL WlL 153. pp. 394-400 (1998)

photoreceptor phytochrome (Hartmann et al., 1983). Two classes of UV-generated mutants with a defect in the phototropic response can be recognised. Class I shows lesions in the biosynthesis of the phytochrome chromophore. Phototropism of these mutants can be rescued with phycocyanobilin and biliverdin (Lamparter et al., 1996, 1997), both substances known to substitute for the phytochrome chromophore in chromophore-deficient mutants of higher plants (Parks and

Somatic Hybridization of Ceratodon

Quail, 1991). Rescue experiments and closer physiological analyses of such mutants have allowed us to identify other phytochrome-mediated effects like control of plastid development and of chlorophyll synthesis. In addition, Class I mutants allow phytochrome-mediated light responses and those mediated by a separate blue light photoreceptor to be distinguished (Lamparter et al., 1997). In Class II mutants, on the other hand, only the phototropic response is lost, other phytochrome-controlled responses being normal (Lamparter et al., 1996). In contrast to the chromophore-deficient mutants, this second class is not rescued by chromophore feeding and indeed displays wildtype levels of spectrallyintact phytochrome; the lesion here is not known but might involve signal transduction. Analysis of Class II mutants has shown that phytochrome affects the gravitropic response (Lamparter et al., 1996). The Ceratodon system has advantages for phytochrome research: 1) the filaments grow well in darkness, 2) mutants can easily be isolated and 3) responses can be analysed at the cellular level. These advantages are in part counterbalanced by the fact that genetic analysis of mutants is restricted because sexual reproduction has not yet been established under laboratory conditions. Although this precludes classical complementation analysis, somatic hybridisation following protoplastfusion and -regeneration provides an alternative way to establish the number of complementation groups involved in one phenotype. This technique has successfully been used for the moss Physcomitrella patens using mutants originating from different auxotropic strains (e.g. Grimsley et al., 1977) or using wildtype and a mutant with an altered chloroplast pattern that can be detected in the fusion product (Rother et al., 1994). Single cell fusion has successfully been reported for protoplasts of the moss Funaria hygrometrica (Meija et al., 1988). This paper describes fusion of Ceratodon protoplasts of two different phototropism mutants and a comparative analysis of this fusion product. Materials and Methods

Moss strains and culture conditions The wildtype strain wt4 and the phototropism mutants ptr103 and ptr 116 have been described (Lamparter et al., 1996 and 1997). The mutant ptrp 19 was isolated from regenerated wt4 protoplasts via its aphototropic phenotype. Filaments were grown on cellophane sheets on solid agar (1.1 %) as described (Lamparter et al., 1996): unless indicated otherwise, BCD medium was used (Cove et al., 1996), consisting of 10 mmollL KN0 3, 1.8 mmollL KH2P04, 1 mmollL MgS0 4, 10 I1mollL C6H5Fe07 and trace elements, pH 6.5 (KOH), to which sterile CaCh and ammonium tartrate, at a final concentration of 2 and 5 mmollL, respectively, were added after autoclaving. Standard growth conditions were 20 'C, and 16 h light (fluorescent tubes Phillips TL65W/25RS, 100 I1mol m -2 s-I PAR)I 8 h dark cycle.

Protoplast-isolation, -fUsion and -regeneration Protoplast-isolation and -regeneration was carried out following Cove et al. (1996): moss tissue was blended with an Ultraturrax and grown under standard conditions for 7 days. The cell wall was di-

395

gested for 30 min at room temperature with 2 % driselase (Sigma) in 8 % mannitol; then the protoplasts were filtered through a 50 11m metal mesh, pelleted for 5 min at 1006'.>, and washed 3 times with 8 % mannitol. For fusion experiments, a variation of the protocol of Grimsley et al. (1977) was used. After adjusting protoplast density to 5 x 105 per mL, protoplasts of two different mutants were mixed at a 1 : 1 ratio and then mixed with 40 % polyethylene glycol (PEG) 3400 (Sigma), and 100 mmollL Ca(N03h. pH 8 (KOH) to give a final PEG concentration of 22 %. After 10 min, protoplasts were washed 3 times with 8 % mannitol. For controls, protoplasts from individual mutants were treated similarly. The protoplasts were allowed to regenerate into filaments on standard growth medium after addition of 8 % mannitol, 2 mmollL CaCh and 5 mmollL ammonium tartrate, overlaid with cellophane. After 3 days on regeneration medium, protoplasts were transferred to the medium that was used for physiological assays (see below). The prolonged 3-day incubation period on mannitol medium resulted in a higher regeneration rate than the standard period of 1 day. For selection of fusion products, the plates were placed in unilateral red light (660nm ± 20nm, 15I1molm-2s-l) for 2 to 7 days; filaments growing towards the light could easily be isolated under a binocular microscope.

Physiological analym and phytochromt mtasurements All physiological analyses were done with dark-adapted filaments. For those analyses the 1 b medium described by Hartmann et aI. (1983) was used, consisting of 1 mmollL KN03, 100l1moi/L CaCI2, 1 mmollL KH2P04, 40l1mollL MgS04' 10 I1mollL 4H5Fe07, 27 mmollL glucose, and trace elements, pH 5.8 (KOH). During dark adaptation, the agar plates were placed vertically so that the negatively gravitropic filaments aligned parallel on the surface of the cellophane. The phototropic response was examined with filaments grown for 5 days in darkness. Following unilateral irradiation for 24h with red light (660nm ± 20nm, 15IJ.ffiolm-2s-l) the resulting angle was measured with a computer-coupled video camera and analysing software (see Lamparter et al., 1997). For analysing the effect of red light on chlorophyll, cells kept for 6 days in darkness were compared with cells grown for 5 days in darkness followed by growth in 24 h red light as above. Chlorophyll fluorescence was analysed in the tip cells using a confocallaserscanning microscope as described by Lamparter et al. (1997). For phytochrome photoreversibility measurements, 500 mg tissue grown for 7 days in darkness were extracted with the use of a French pressure cell and then processed as described (Lamparter et al., 1996). Difference measurements were made in the presence of CaC03 as scattering agent with a computer-controlled dual-wavelength spectrophotometer as described by Lamparter et al. (1994).

Flow cytomttry Moss tissue was grown as described above for the isolation of protoplasts, but the period following inoculation was extended to 14 days. For comparison, freshly picked leaves of 14-day-old ArabU/opsis cv. Landsberg erecta were used. Extraction of nuclei and flow cytometry followed Samoylova et al. (1996). About 200 mg tissue were chopped in a glass Petri dish with a new razor blade in 2 mL of DAPI-containing buffer (Partee, Germany) for ca. 5 min until a homogenous slurry was achieved. Thereafter, nuclei were separated from debris by filtering through a 50 11m nylon mesh. Nuclei were incubated for 24 h at 4 ·C to achieve complete saturation of the DAPI stain. DAPI fluorescence, and forward and lateral scatter of particles were measured in a flow Cytometer (FACStar PLUS, Becton Dickinson. San Jose. CA. USA) equipped with an argon ion laser (INNOVA 905 5 W. Coherent, Palo Alto. CA, USA). Each measurement was based on fluorescence intensities of 10,000 particles.

396

T. !.AMPARTER, G. BROCKER, H. EsCH, J. HUGHES, A. MEISTER, and E. HARTMANN

Subsequently, the fluorescence data were gated according to the associated forward-scatter in order to eliminate signals arising from cell debris.

Results

Table 1: Results from flow cytometry measurements with DAP I stained nuclei of Arabidopsis thaJiana and Cmztodon purpureus as presented in Fig. 1. Arabidopsis nuclei were measured 5 x, Ceratodon wt4 5x, ptr103 3x, ptr1162 x, ptr103(+ )ptr1163 X and ptrp192x. Of each strain, the measurements with the lowest CV value was taken. Shown are the mean values of the GI and G2 peaks ± CV as calculated by the flow cytometer sofrware.

Protoplast fosion For protoplast isolation we followed Cove et al. (1996), with modifications as described in Materials and Methods. The major variation concerned the duration of incubation on mannitol-containing medium: protoplasts were kept for 3 days instead of 1 day on this medium, because the rate of regeneration was higher. Under our conditions, the yield was typically around 2 X 107 protoplasts per g fresh weight and regeneration rates were around 30 to 50 %. Following PEG-mediated fusion of protoplasts, the regenerated filaments were grown on vertically-oriented plates for up to 7 days in continuous unilateral red light given horizontally, parallel to the surface of the agar medium. Under these conditions, while wildtype filaments grew positively phototropically (towards the light), those of ptrl03 grew in random directions, whereas those of ptr116 grew negatively gravitropically (Lamparter et al., 1996, 1997). Since the three strains are thus distinguishable from each other, fusion products of mutants complementing each other were easily identified. Indeed, in experiments where ptr 103 and ptr116 protoplasts were subjected to the fusion procedure, positively phototropic filaments were identified with a frequency of 18-26 per plate (4 independent experiments, around 50,000 regenerated filaments per plate). Such wildtype-like filaments were never found if ptr103 or ptr116 alone were subjected separately to the fusion procedure (for each mutant 4 experiments done in parallel). The fusion rate of ptr103 and ptr116 was not improved by increasing the PEG-concentration in contrast to Rother et al. (1994). The phenotype of numerous putative fusion products was indistinguishable. For flow cytometry and physiological characterization we focussed on an arbitrarilychosen line, preliminarily named ptr103( +)ptr116.

Plow cytometry of Ceratodon To see whether the positive phototropic phenotype of

ptr103( +)ptr116 might result from protoplast fusion fol-

Fluores- Fluores- Ratio G2 (c. p.)1 cence of cence of G2/G 1 G2 (A. t.)

G 1 peak G2 peak

Arabidopsis Ceratodon wt4

86± 10 104± 9 ptr103 114±15 ptr116 124± 13 ptr103 ( +) ptr116 209± 5 ptrp19 115± 5

180±6 219±4 241±5 243±3 426±3 236±3

2.08 2.1 2.13 1.97 2.04 2.06

1.22 1.34 1.35 2.37 1.31

tion; the histogram pattern resulting was similar to the sum of both single extractions (data not shown). The lowest intensity fluorescence peak with wildtype Ceratodon was around channel No. 104, the second at 219 and the third at 430. In all cases the fluorescence intensity of the first peak was almost exactly half that of the second. Although in the wildtype the first peak was small (few nuclei with this DNA content), it was always present. The phototropism mutant, ptrp 19, on the other hand, showed a much larger first peak, positioned identically. The peak positions of 1 C and 2 C of ptr103, ptr 116 and ptrp 19 corresponded to slightly stronger fluorescence signals than those seen in the wildtype, whereas nuclei of the putative fusion product ptr 103( +)ptr116 gave fluorescence peaks almost exactly twice that of the wildtype. In initial studies a second fusion product was also analysed, yielding results (not shown) similar to those for ptr 103( +)ptr 116 given here.

Physiology ofptrl03{+ )ptr116 Results of experiments with red-light induced phototropism of wildtype and the mutant strains are summarised in Table 2. Under those conditions the wildtype curvature of 82° is close to the maximally possible 90° response. The 32° curvature of ptr 103 in these experiments is greater than the 8° reported earlier (Lamparter et al., 1996), resulting from the increased light intensity used. During prolonged red light irradiation (over a period of 5 days) the filaments of this mu-

lowed by fusion of the nuclei of each mutant, we analysed the nuclear DNA content of this strain in comparison to the wildtype and the mutants from which it was derived. The expected genome size for ptr 103( +)ptr116 would be twice that of the wildtype, ptr 103 or ptr116. This analysis was done by Table 2: Phototropic response of Cmztodon wildtype and different measuring fluorescence of DAPI-stained nuclei with flow cy- mutants. Mean values ± SE of three or more experiments each including at least 30 fIlaments. tometry. Such measurements also provide an estimate of the Ceratodon genome size, previously unknown, using nuclei of dark after 24h young leaves of Arabidopsis (1 C = 100 Mbp) as a reference. control unilateral red light Each strain was measured several times (see legend of Table OO± 1° 82°± I· 1), always yielding the same qualitative result. Histograms of wildtype 31°±2° n.d. the measurements are presented in Fig. 1 and summarised in ptr103 0·±1° 0·±1° Table 1. Three peaks were apparent in data from both Arabi- ptr116 66°±2· ptr1160n 0.25 mmol/L biliverdin n.d. dopsis (Fig. lA) and Ceratodon (Fig. 1 B-E). In one experi- ptr103 ( +) ptr116 OO±I· 79°±1° ment, Arabidopsis and Ceratodon were mixed prior to extrac-

Somatic Hybridization of Ceratodon

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Table 3: Chlorophyll fluorescence of dark-adapted and red-lighttreated Ctratodo" strains. Relative units.

mixed under appropriate conditions and a putative somatic fusion product ptr 103( + )ptr116 isolated via its wildtype-like phenotype. The genome sizes of wildtype, mutant and fusion 6 days 5 days darkness, product were measured alongside that of Arabidopsis thaliana darkness 1 day red light by flow cytometry. Phototropism and chlorophyll content of tip cells as well as phytochrome levels of dark-adapted fila60±8 159±12 wildtype ptr103 28±3 91±8 ments were also measured. ptr1l6 3±0.8 1.3±0.3 Using the work of Grimsley et al. (1977) with Physcomiptr103( +) ptr1l6 21±4 105±8 trella patens as a guide, we established protoplast fusion and regeneration for Ceratodon purpureus. This was possible because under long term irradiation both mutants could easily Table 4: Phytochrome photorcversibility of dark-adapted Ctratodo" be distinguished from each other and from the wildtype (see strains. Mean values ± SE of 4 extractions of moss tissue kept for 7 Introduction). Auxotrophic strains have been used for protodays in darkness. plast fusion of physcomitrella mutants (e.g. Grimsley et al., 1977), fusion products being identified by their growth under 10- 3 MAI(g fw) minimal medium conditions. Ceratodon auxotrophs are not available, but we are presendy raising phototropism mutants 20±1 wildtype from different antibiotic-resistant strains, allowing comple16±1 ptr103 mentation testing of phenotypically-similar mutants. With ptr1l6 0±1 ptr103(+)ptrl16 12±1 the procedure used here the rate for heterologous fusion is estimated to 0.05 % (around 25 in about 50,000 protoplasts). This frequency is rather low compared with results obtained et al., 1977; Rother et al., 1994). tant grew randomly. Although this property is difficult to for Physcomitrella (Grimsley 2 + and PEG concentrations did not sigHowever, varying Ca quantify, it allows ptr103, ptr116 and wildtype to be distinguished easily. ptr116 showed no phototropic response: nei- nificandy increase the number of fusion products. The low ther a 24 h unilateral irradiation (Table 2) nor prolonged irra- efficiency might result from the ptr116 protoplasts themdiation (data not shown) elicited significant bending. Biliver- selves: This mutant with a defect in bilin synthesis and low din feeding experiments (Table 2) confirm that the phototro- chlorophyll content produces additional cell wall compic response of ptr116 can be rescued with this tetrapyrrole. ponents that might act inhibitorily on fusion with other proThe fusion product ptr 103( +)ptr116 showed a response clo- toplasts. The flow cytometric data for Arabidopsis (Table 1 & Fig. 1) sely similar to that of the wildtype. Previous studies have shown that phytochrome controls are in accord with published results (Samoylova et al., 1996). chlorophyll accumulation in Cn-atodon. Changes in chloro- The peak with the lowest fluorescence corresponds to 2 C (2 phyll content of the tip cell can be analysed at the cellular copies of each chromosome; diploid nuclei in G 1), the seclevel by quantifying images from confocal laserscanning ond peak to 4 C (4 copies; diploid nuclei in G 2 or tetraploid microscopy (Lamparter et al., 1997). Measurements were nuclei in G 1). Higher C-values as implied by the third peak again carried out with wt4, mutants and the fusion product, are commonly found in leaves of Arabidopsis and indicate powith results summarised in Table 3. A red-light-induced in- lyploidisation of cells. In contrast to sporophytic Arabidopsis crease of chlorophyll fluorescence was seen in the wildtype; leaf cells, Ceratodon filament cells are gametophytic and smaller changes were seen in ptr 103 and ptr 103 (+ ) ptr 116, hence presumably haploid. We interpret the first peak from Ceratodon as 1 C, corresponding to haploid nuclei in the G 1 whereas for ptr116 there was no significant change. phase. The second peak, which is always found at fluorescence intensities of double that of the first peak, is thus interPhytochrome spectral measumnmts preted as 2 C. The large area below this peak indicates that We also measured phytochrome content of the different most nuclei are in this condition. As most of the cells anastrains after dark adaptation (Table 4). Phytochrome content lysed have stopped dividing, our interpretation of this peak is in wildtype protonemata was down-regulated in the light; fil- not certain: the double DNA content might result from prior aments placed in darkness slowly accumulated phytochrome DNA replication either during the cell cycle (i.e. this populaover a 7-day period (Lamparter et al., 1995). The values for tion is arrested in G 2) or from polyploidisation (i.e. diploid ptr103 and ptr116 were in accord with earlier findings, in G 1). The former assumption has been made for the leafy ptr103 showing phytochrome photoreversibility similar to the moss Physcomitrella patens (Reski et al., 1994). However, for wildtype and ptr116 containing almost no spectrally-active seed plants and indeed most eukaryotes, the arrest of the cell phytochrome. The phytochrome content of ptr 103( +) ptr 116 cycle occurs in Gland a high G 2/G 1 ratio is only found in tissue with a high cell division rate (see Galbraith et al., was intermediate. 1983). The alternative explanation for Ceratodon would suggest that most of the cells have undergone polyploidisation to the diploid stage. Flow cytometry data for the mutant Dfscusslon ptrp 19 (a spontaneous mutant) showed almost equal first and In this study, protoplasts from two different phototropism second peaks (Fig. 1); either this mutant has an altered cell mutants of Ctratodon purpureus, ptr 103 and ptr116, were cycle or the degree of polyploidisation is lower. The data

Somatic Hybridization of Ceratodon

presented in Fig. 1 were made 3 months after isolation of

ptrp 19; additional measurements done 3 months later

399

(Lamparter et al., 1997) and is certainly a result of the ptr 116 mutation. Phototropism studies and measurement of phytochrome photoreversibility were also consistent with the notion that the two mutants, ptr 103 and ptr 116, complement each other in the wildtype-like somatic fusion product ptr 103( +)ptr116. Protoplast fusion is a powerful tool in complementation analysis. This is the first report of the technique in Ceratodon. Strains marked with different antibioticresistance genes will now allow complementation tests with mutants that are phenotypically indistinguishable. A further advantage of Ceratodon in various genetic contexts is its unusually small genome, as revealed in this work.

revealed a larger second peak, a pattern similar to that for the wildtype (data not shown). It is possible that ptrp19 was isolated as a haploid line and diploidisation occurred during subsequent cultivation. Data for the UV-induced mutants ptr 103 and ptr116 implied a slighdy (ca. 10 %) but consistently larger genome size than in wildtype and ptrp 19. Although UV mutagenesis is thought usually to result in point mutations in bacteria, this might not be the case in plants (Britt, 1995). Nevertheless, it seems unlikely that ptr 103 and ptr116 indeed have larger genomes than the wildtype as the fusion ptr103(+)ptr116 showed exactly twice the genome size of the wildtype. The Acknowledgements origin of this discrepancy is as yet unknown. Quantitative comparison of the flow cytometric data from We thank S. Misera (IPK Gatersleben) for his help with the flow Arabidopsis and wildtype Ceratodon allows their relative gen- cytometry. ome sizes to be estimated. The Arabidopsis genome size has been extensively analysed using various methods, yielding values between 70 and 150 Mbp for 1 C (Meyerowitz, 1994). References The fluorochrome DAPI that has been used for our measurements is an AfT specific dye. Leutwiler et al. (1984) derived BENNETT, M. D. and J. B. SMITH: Nuclear DNA amounts in anan AT-content of 59 % for the Arabidopsis genome whereas giosperms. Phil. Trans. R. Soc. Lond. B 274, 227-ZJ4 (1976). Reski et al. (1994) gave a value of 65 % for that of Physcomi- BRITT, A. B.: Repair of DNA damage induced by ultraviolet radiatrella. There is no equivalent data for Ceratodon itself The AT tion. Plant Physiol. 108, 891-896 (1995). content of published Ceratodon gene sequences is 58 %, close COVE, D. J., R. S. QUATRANO, and E. HARTMANN: The alignment to the 56 % derived from the homologous gene sequences in of the axis of asymmetry in regenerating protoplasts of the moss, Arabidopsis. The published gene sequences of Physcomitrel/a Ceratodon purpureus, is determined independently of axis polagive a mean AT-content of 53 %; the large pumilio gene in rity. Development 122,371-379 (1996). the same species yields 52 % (personal communication, A C. GALBRAITH, D. w., K. R. lliIuaNS, J. M. MADDOX, J. M. AYRES, D. P. SHARMA, and E. FIRooZABADY: Rapid flow cytometric analCuming, University of Leeds). The origin of the high value of ysis of the cell cycle in intact plant tissues. Science 220, 1049Reski et al. (1994) for this species might be extensive, non1051 (1983). coding AT-rich genomic regions. The values for 1 C Ceratodon ranged between 1.22 X and 1.35 x those of 2 C (G 11 GOODMAN, H. M., J. R. EcKER, and C. DEAN: The genome of Arabidopsis thaliana. Proc. Natl. Acad. Sci. USA 92, 10831-10835 diploid) Arabidopsis (Table 1): assuming an Arabidopsis gen(1995). ome size of 100 Mbp (Goodman et al., 1995) and a similar AT content, the genome size for Ceratodon is about 250 Mbp. GRIMSLEY, N. H., N. W. AsHTON, and D. C. COVE: Complementation analysis of auxotrophic mutants of the moss, Physcomitrella Genome sizes of other seed plants (Bennett and Smith, 1976; patens. Mol. Gen. Genet. 154,97-100 (1977). Rogers and Bendich, 1988) and also of other mosses includHARTMANN, E., B. KLINGENBERG, and L. BAUER: Phytochrome meing Physcomitrella patens (unpublished data; Reski et al., 1994 diated phototropism in protonemata of the moss Ceratodon purand personal communication) are all significantly larger. pureus BRID. Photochem. Photobiol. 38, 599-603 (1983). Thus, we consider Ceratodon to have the second smallest gen- l..AMPARTER, T., J. HUGHES, and E. HARTMANN: A fully automated ome known for plants. dual-wavelength photometer for phytochrome measurements and Light regulated tip-cell chlorophyll levels were investigated its application to phytochrome from chlorophyll-containing exusing in situ fluorescence microscopy (Table 3). Red light tracts. Photochem. Photobiol. 60, 179-183 (1994). induced an increase in the wildtype, ptr 103 and ptr 103( +) l..AMPARTER, T., S. PODWWSKl, F. MITrMANN, H. J. SCHNEIDERPOETSCH, E. HARTMANN, and J. HUGHES: Phytochrome from ptr116, whereas in ptr116there was no significant change. This protonemal tissue of the moss Ceratodon purpureus. J. Plant Physindicates that the phytochrome regulation of chlorophyll iol. 147, 426-434 (1995). accumulation is almost normal in ptr 103 and ptr 103( +) ptr116. The values for ptr103 and ptr103(+)ptr116 were l..AMPARTER, T., H. EsCH, D. COVE, J. HUGHES, and E. HARTMANN: Aphototropic mutants of the moss Ceratodon purpureus: Lines nevertheless lower than for the wildtype. It is known that with spectrally normal and with spectrally dysfunctional phytowith increasing period of dark growth the chlorophyll fluochrome. Plant Cell Environm. 19,560-56 (1996). rescence of tip cells drops steadily with a steep decay during l..AMPARTER, T., H. EsCH, D. COVE, and E. HARTMANN: Phytothe first 12 days of dark growth (Lamparter et al., 1997). Difchrome control of phototropism and chlorophyll accumulation ferences found between different strains after 5 days of dark in the apical cells of protonemal filaments of wild-type and an incubation (the duration used in this paper) may also result aphototropic mutant of the moss Ceratodon purpureus. Plant and from subtle differences in growth conditions or from the hisCell Physiol. 38, 51-58 (1997). tory of each culture. The very low chlorophyll fluorescence of MEJIA, A., G. SPANGENBERG, H.-U. Koop, and M. Bopp: Microculdark-grown ptr 116, however, which was in this case 7- to 20ture and electro fusion of defined protoplasts of the moss Funaria hygrometrica. Bot. Acta 101, 166-173 (1988). fold lower than of any other strain, confirms earlier findings

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ROGERS, S. O. and A. j. BENDICH: Extraction of DNA from plant tissues. Plant Mol. BioI. Manual A6, 1-10 (1988). ROTHER, S., B. HADELER, j. M. ORSINI, W. O. ABEL, and R RESKI: Fate of a mutant macrochloroplast in somatic hybrids. j. Plant Physiol. 143,72-77 (1994). SAMOYLOVA, T. 1., A. MEISTER, and S. MISERA: The flow karyotype of Arabidopsis thaliana interphase chromosomes. Plant Journal 10, 949-954 (1996). SCHWUCHOW, j., F. D. SACK, and E. HARTMANN: Mictotubule distribution in gravitropic protonemata of the moss Ceratodon. Protoplasma 159,60-69 (1990).