Phytochrome Control of Phototropism and Chlorophyll

Plant Cell Physiol. 38(1): 51-58 (1997) JSPP © 1997

Phytochrome Control of Phototropism and Chlorophyll Accumulation in the Apical Cells of Protonemal Filaments of Wildtype and an Aphototropic Mutant of the Moss Ceratodon purpureus Tilman Lamparter1, Heike Esch 1 , David Cove 2 and Elmar Hartmann' 1 2

Institute for Plant Physiology, Free University Berlin, Konigin Luise Str. 12-16, D-14195 Berlin, Germany Department of Genetics, University of Leeds, Leeds LS2 9JT, U.K.

Key words: Blue light photoreceptor — Ceratodon purpureus — Phototropism — Phycocyanobilin — Phytochrome (chromophore) — Regulation of chlorophyll synthesis.

Abbreviations: /dJ A, red/far-red reversible change of absorbance difference between two wavelengths; n.d., not determined; PCB, phycocyanobilin; Pfr, far-red absorbing form of phytochrome; Pr, red absorbing form of phytochrome; s.e., standard error of the mean. 51

Phototropism of the protonemal apical cell of the moss Ceratodon purpureus is controlled by the plant photoreceptor phytochrome (Hartmann et al. 1983). Following UV mutagenesis, several aphototropic lines have been isolated that are apparently defective in the biosynthesis of the phytochrome chromophore (Lamparter et al. 1996). In such mutants all phytochrome responses are expected to be defective and a comparison with wildtype lines may point to other physiological effects in addition to phototropism that are under the control of phytochrome. Such a comparison has already shown that the gravitropic response is down-regulated by phytochrome (Lamparter et al. 1996), and an identification of further phytochrome-controlled responses may help to establish C. purpureus as a model system for phytochrome research. Since light-grown tissue of ptrl, the mutant characterized initially, shows significantly lower chlorophyll levels than the corresponding wildtype strain, phytochrome may have a regulatory role in chloroplast development and/or chlorophyll biosynthesis. The regulation of these processes is well analyzed during de-etiolation of angiosperm seedlings. Two different light-dependent steps are involved in the development of fully intact green chloroplasts. One step requires the activation of the photoreceptors phytochrome and the blue light receptor. The other step is the conversion of protochlorophyllide into chlorophyllide, which is dependent on light absorbance via protochlorophyllide (Virgin and Egneus 1983). It is difficult to dissect these two light absorbing systems in angiosperms and to analyze the function of phytochrome separately. Often the effect of phytochrome on chlorophyll accumulation is monitored by estimating the duration of the lag phase of chlorophyll biosynthesis which occurs when etiolated plants are brought into white light. Protochlorophyllide-chlorophyll conversion in mosses is not light dependent and occurs in darkness, allowing a more straightforward assessment of photoreceptor action. A further advantage is that chlorophyll levels can be analyzed in a single growing cell, the protonemal apical cell. For C. purpureus filaments, an effect of light on chlorophyll content and on plastid morphogenesis (Valanne 1971) implies the participation of phytochrome in these processes. However, the roles of phytochrome and of the blue light photoreceptors are not yet clear. We have therefore

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The aphototropic mutant line ptrll6 of the moss Ceratodon purpureus shows characteristics of a deficiency in the phytochrome chromophore. Photoreversibility measurements indicate an approximately 20 time lower concentration of spectrally active phytochrome compared to wildtype, whereas normal phytochrome apoprotein levels are found on immunoblots. Feeding with the tetrapyrroles biliverdin, the proposed precursor of the phytochrome chromophore, or phycocyanobilin, which may replace the phytochrome chromophore, resulted in the rescue of ptrll6 phototropism. The ptrll6 mutant and the phenotypically-related mutant ptrl contain lower chlorophyll levels than the wildtype. Chlorophyll content of wildtype and mutant tissue grown under different light conditions was estimated using conventional spectrophotometry of extracts and fluorimetrically, on single apical cells. Dark-grown tissue contained about 100 times less chlorophyll than tissue grown under standard white light conditions. Red light given for 24 h to dark adapted filaments induced an increase in the chlorophyll content in the wildtype, but not in ptrl16. Blue light induced an increase in chlorophyll both in wildtype and in ptrl 16. The red light effect on the wildtype was partially reversible with far-red, \iptrll6 was grown on phycocyanobilin, an increase in chlorophyll was also found when cells were irradiated with red light. The results indicate that phytochrome as well as a blue light photoreceptor regulate chlorophyll accumulation in C. purpureus protonemata. It can be assumed that in ptrll6, the synthesis of the phytochrome chromophore is blocked specifically beyond the synthesis common to chlorophyll and the phytochrome chromophore and affects an enzymatic step between protoporphyrin and biliverdin.

52

Phytochrome control of chlorophyll biosynthesis

begun to quantify chlorophyll levels from tissue pre-irradiated with different light qualities, using a fluorimetric approach which allows us to follow chlorophyll levels in single protonemal apical cells. This has in turn allowed us to observe the ability of a newly-isolated mutant, which apparently lacks the phytochrome chromophore, to recover chlorophyll biosynthesis after feeding with PCB, suggesting that PCB can replace for the phytochrome chromophore. The same mutant has also allowed us to distinguish clearly between blue light and red light induced processes. Materials and Methods


Moss strains and cultivation—For all analyses, the wildtype wt4 (Hartmann et al. 1983) and an aphototropic mutant, ptrll6, derived from the same strain, were used. ptrll6 was isolated during a screen for aphototropic growth following UV-mutagenesis as described by Lamparter et al. (1996). Filaments were grown on solid lb medium (1 mM KNO3, 100 /xM CaCl2, 1 mM KH2PO4, 10 nM C6H3FeO7, 27 mM glucose, trace elements, adjusted to pH 5.8 with KOH, and 1.1% Agar (Sigma) (see Lamparter et al. 1996)). Standard growth conditions were: 20°C in a 16 h light (fluorescent tubes Philips MCFE white; fluence rate lOO/rniolm"2 s~' PAR)/8 h dark cycle. For dark adaptation filaments were grown in black boxes on cellophane overlaying agar medium at 20° C. The agar plates were always placed vertically so that the apical cells aligned parallel on the surface of the cellophane, as a result of their negative gravitropism. Tetrapyrrole feeding—Stock solutions of tetrapyrroles were made as follows. Biliverdin dihydrochloride (Sigma) was dissolved in water at a final concentration of 0.5 mM by continuous stirring, adjusted to pH 6.0 with KOH. Phycocyanobilin (PCB) was prepared from the blue alga Spirulina geitlerie according to Kunkel et al. (1993), dissolved with dimethyl sulfoxide to give a final concentration of 2 mM, as monitored spectrophotometrically using the molar absorption coefficient of 37,900 M" 1 cm" 1 at 680 run, diluted 1/100 with water, and adjusted to pH 6.0 with KOH. Protoporphyrin IX disodium salt (Sigma) was dissolved in water to a final concentration of 2 mM, adjusted to pH 7.5 with HC1. The more alkaline pH was necessary to yield soluble protoporphyrin. Heme (protoheme, Sigma) was prepared in the same way as described for protoporphyrin. Stock solutions were sterilized by filtration and then added to equal quantities of melted, doublestrength lb medium at 50°C, to give final concentrations of tetrapyrroles of 0.25 mM for biliverdin, lOjuMforPCB, 1 mM for protoporphyrin and heme. Twenty four hours prior to physiological assays, the cellophane carrying dark-adapted filaments was transferred to tetrapyrrole-containing medium. Filaments used for controls were transferred to tetrapyrrole-free medium. Phytochrome measurements and immunoblotting—Protonemal tissue was extracted using a French pressure cell (Mini-Cell FA003, SLM Instruments, Rochester, NY, U.S.A.) and processed as described previously (Lamparter et al. 1995, 1996). Photoreversibility was measured in a computer-controlled dual wavelength photometer in which the measuring wavelengths are set to 670 and 780 nm (Lamparter et al. 1994). Actinic irradiation occurred at 660 ± 12 nm and broadband far-red above 725 nm (RG-9 filter, Schott, Mainz, Germany). Spectral assays utilised 10 mm diameter cuvettes containing 400 fil extract mixed with 250 mg CaCO 3 , a scattering agent enhancing the photoreversibility signal by extending the light path.

Immunoblots were prepared as described previously (Lamparter et al. 1995) using SDS-PAGE (1% separating gel). Phytochrome was immunostained with affinity-purified APC1 polyclonal antibody (Lamparter et al. 1995). Phototropic response—The phototropic curvature was estimated from filaments that have been aligned by negative gravitropic growth on cellophane-overlayed, vertically oriented plates. After 5 d growth in darkness, phototropism was induced using red light from a halogen projector applied through a 665 + 12 nm DAL interference filter (Schott, Mainz, F.R.G.) at an intensity of 4/jmolm~ 2 s~' for 24 h. Light was given horizontally, parallel to the agar surface, so that a maximal phototropic response would give a 90° deviation from the original growth direction. The resulting angle was evaluated using a microscope, a computer-coupled video camera and an imaging software program (Image P2, H + H MeBsysteme, Berlin, Germany). Chlorophyll extraction and measurement—Light-grown tissue was taken directly from moss cultures grown for 7 d after subculturing under standard light conditions. Dark-adapted tissue was obtained by growing filaments at 20° for 14 d on vertically-oriented agar plates in darkness. Red and far-red irradiations were given using the apparatus described for phototropism analyses with 665 ± 12 nm (red) or 735 ± 12 nm (far red) DAL interference filters. Light intensities were 4/imol m~2 s~' for red and 5 ^mol m~2 s" 1 for far-red. The filaments grown during the dark incubation period were carefully separated from the older tissue below by cutting with a sharp razor blade. Routinely, 50 mg fresh weight were extracted with 1 ml of 80% acetone by incubating at 4° in darkness for 4 h. The filaments were separated from the supernatant by centrifugation (48,000 x g, 15 min) and absorbance was measured using a Kontron (Neufahrn, Germany) 941 spectrophotometer at 700, 663, 652 and 645 nm with an integration time of 30 s for each wavelength. The 700 nm absorbance was taken as an internal standard and subtracted from the values obtained for 663, 652 and 645 nm. The concentration of total chlorophyll was obtained from the sum over chlorophyll a and b as calculated by the formulae given by Arnon (1949) and was routinely compared with the value for total chlorophyll estimated from the isosbestic point 652 nm. Spectra were recorded at 100 nm per min using an 80% acetone baseline. For low absorbing samples, averages were taken from three spectra recorded from the same sample. Chlorophyll fluorescence imaging and quantification—Moss filaments were grown either under standard white-light conditions or on vertically-oriented plates in darkness for 5 d and thereafter irradiated for 24 h with different light programs as described above. For quantification of chlorophyll fluorescence, specimens were imaged using a confocal laser-scanning microscope consisting of an Axiowert 35 with inverted optics and a 40x PlanNeofluar objective (Zeiss, Oberkochen, Germany) coupled to a MRC 1024 laserscan system (Biorad, Hemel Hempstead, Great Britain). The 650 nm red band of the krypton-argon laser was always selected for excitation; a 680 nm cutoff filter was selected for the emission light path. Apical cells were selected arbitrarily through the normal optics of the microscope. Cells that were not'in contact with other cells were chosen. For all measuring procedures, the high voltage of the photomultiplier was set to 1,000 V and the aperture was held maximally open (8.0). To adapt the system to the greatly varying fluorescence signals, the intensity of the excitation beam was adjusted via neutral density filters in order to generate signals just below the saturation level. Control experiments have shown that if


Results Phototropism and phytochrome content of the aphototropic mutant, ptrl!6—The newly isolated aphototropic mutant ptr!16 is derived from the wildtype strain wt4, while the mutant ptrl, that was analyzed previously is derived from the wildtype strain wt3. As wt3 shows a reduced phototropic response compared to wt4 (Lamparter et al. 1996), the difference between mutant and the corresponding wildtype with respect to phototropism is higher for ptrl 16 than for ptrl. Table 1 shows that during a 24 h unilateral red light irradiation, no phototropic bending was induced in ptrl 16. Under those conditions the wildtype showed an 85° response. Because p/r/7 6 was phenotypically similar to ptrl, which had been identified as a mutant lacking the phytochrome chromophore (Lamparter et al. 1996), the recovery of ptrl 16 phototropism was tested with the four tetrapyrroles protoporphyrin, heme, biliverdin and phycocyanobilin. In higher plants, it is proposed that the biosynthesis of the phytochrome chromophore, phytochromobilin, follows the synthesis of chlorophyll and branches at the position of protoporphyrin (see Weller et al. 1996). It is sug-

Table 2 Phytochrome photoreversibility of dark adapted wildtype and ptrl 16 FW) wt4

8.8±0.4

ptrll6

0.3±0.2

gested that heme is formed via Fe 2+ chelation, which is transformed into biliverdin and finally phytochromobilin. A very weak positive phototropic response of around 5° (Table 1) was observed if ptrl 16 was grown on protoporphyrin (1 mM) or heme (1 mM). Both biliverdin (0.25 mM) and PCB (10 /uM), which may replace the phytochrome chromophore, rescued the phototropism of ptrl 16 almost totally; in both cases the bending curvature was around 60°. The phototropic response of wt4 was not affected by PCB (Table 1). Extracts of ptrl 16 contained very low levels of spectrally-active phytochrome (Table 2). The value for ptrl 16 was around the detection limit of the measuring instrument, and at least 20 times lower than the wildtype value. On immunoblots, the anti-phytochrome-antibody APC1 stained an apophytochrome band in wt4 and in ptrl 16 (Fig. 1). Chlorophyll content of light and dark-grown tissue— The chlorophyll content of ptrl 16, grown under standard white light conditions, was about 5 times lower than the content found in wildtype (Table 3). In both strains, darkgrown filaments contained lower chlorophyll levels than filaments grown in white light. After a 2 week dark adaptation, both wildtype and mutant contained a similar low amount of chlorophyll, around 5//g per g fresh weight (Table 3). An example of absorbance spectra is shown in Fig. 2. The extract of dark-grown tissue still exhibited an absorbance maximum at 663 nm characteristic of chlorophyll a. Similar spectra were obtained from filaments that were grown for 4 weeks in darkness. A constant 24 h red light irradiation given to dark-

Table 1 Phototropic response of wildtype and ptrl 16 Standard medium wt4

ptrll6

Curvature 0 Protoporphyrin Heme

Biliverdin

PCB

85±1

n.d.

n.d.

n.d.

84±1

0±l

5±2

6±2

62±3

57±2

Filaments were dark adapted for 5 days and then irradiated with unilateral red light for 24 h. Prior to phototropic stimulation, filaments were transferred to protoporphyrin (1 mM), heme (1 mM), biliverdin (0.25 mM) or PCB (10//M). Mean values±standard error of 100 or more cells.


several images are taken from one cell at different excitation intensities, the subsequent quantification yielded the same result within an error of ± 5 % . For each specimen, fluorescence images of 10 or more cells were stored on hard disk. For each cell a relative value for the fluorescence intensity within the area of the most apical 100/im was estimated using the imaging software Lasersharp 1.01 (Biorad). This value, divided by 1,000 and divided by the intensity of the exciting light beam in % is given as "fluorescence units". For the result of a single experiment the average value of the readings from these single cell images was calculated. From 4 or more single experiments the average and the standard error were calculated. Calibration of fluorescence was done with protoplasts, prepared according to Cove et al. (1996), washed and concentrated by 100 x g centrifugation. Chlorophyll concentration of the preparations was measured spectrophotometrically as above and fluorescence signals of defined volumes were quantified under same conditions as for C. purpureus cells. Dilution series showed linearity up to 0.5 ng chlorophyll in a volume of 5 nl, the highest concentration tested (r 2 =0.977). One fluorescence unit was equivalent to 0.04 pg chlorophyll.

53

54


B

0,70

0,030

0,60

i

0,025 light grown

•

0.50

dark grown

0,020

~

0,40

0,015 0,30

0,010 0,20