Isolation of nuclear, chloroplast and mitochondrial DNA from the moss Physcomitrella patens

Plant Science, 61 (1989) 235-- 244

235

Elsevier Scientific Publishers Ireland Ltd.

ISOLATION OF NUCLEAR, C H L O R O P L A S T AND M I T O C H O N D R I A L DNA FROM T H E MOSS PHYSCOMITRELLA P A T E N S ~

JOACHIM R. MARIENFELD, RALF RESKI, CHRISTIANE FRIESE and WOLFGANG 0. ABEL**

Institut fiir AUgemeine Botanil¢ Arbeitsbereich Genetil¢ Ohnhorststrasse 18, D-~OOOHamburg 5~ lF.R.GJ {Received October 1st, 1988} (Revision received December 13th, 1988) (Accepted December 13th, 1988) In this report efficient, rapid and easy methods for the isolation of nuclei, chloroplasts and mitochondria and their corresponding DNAs from a large scale culture of the moss Physcomitrella patens are described. Microscopical controls of organelle preparations revealed single and intact nuclei, chloroplasts and mitochondria. Per 10 g freshweight average yields of 15 ~g nuclear DNA, 4 ~g chloroplast DNA and 1 ~g mitochondrial DNA were obtained. The purity of organelle DNA was tested by hybridization with non-corresponding organelle specific clones. Isolation of nuclei and mitochondria from mosses and characterization of their DNAs are presented for the first time.

Key words: nuclear DNA; chloroplast DNA; mitochondrial DNA; moss; Physcomitrellapatens

Introduction

Isolated plant organelles are considered a good source for the isolation of highly purified organelle DNA. Several methods for isolating nuclei [ 1 - 3 ; for review see Ref. 4], chloroplasts [5,6; for review see Ref. 7], and mitochondria [9-11; for review see Ref. 12] have been established. Although a restriction and gene map of the ptDNA of the moss PhyscomitreUa patens has been published recently [5,6] there is a lack of data concerning the isolation of organelles and the characterization of their respective DNA from mosses. Among other bryophytes chloroplast DNA (ptDNA) has only been characterized in the liverworts Sphaerocarpos doneUii [13] and Marchantia polymorpha [14]. Recently *This article is in part based on doctoral studies by J.R.M. and R.R. in the Faculty of Biology, University of Hamburg. **To whom all correspondence should be addressed. Abbreviations: BSA, bovine serum albumine; EDTA, ethylenediaminetetraacetic acid; MES, [2-(N-Morpbolino)ethanesulfonic acid]; MOPS, [3-(N-Morpholino)propanesulfonic acidl; PVP, polyvinylpyrrolidone; SDS, sodium dodecylsulfate.

the complete ptDNA sequence of the liverwort Marchantia polymorpha has been published [15]. In general large amounts of plant material (50--500 g} are required for isolation procedures and it is inevitable to culture large amounts of protonemata. The methods for organelle isolation then have to be adapted to the special conditions in mosses. In this paper we communicate easy, efficient and rapid methods for large scale culture of moss protonema and the isolation of organelles and organelle DNA from the moss Physcomitrella patens. For the first time methods for isolating nuclei and mitochondria and for the characterization of nuclear DNA (nucDNA) and mitochondrial DNA (mtDNA) from Physcomitrellapatens are described. Materials and methods

Plant material and culture conditions Moss protonemata of Physcomitrella patens were grown axenically at 25 °C under a light-/ dark regime (a combination of Osram Groluxand White-Light lamps} of 16/8 h in a modified

0168-9452/89/$03.50 © 1989 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland

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Knop medium [16] in 500 ml Erlenmeyer flasks capped with silicone sponges (Bellco, Vineland). After an incubation period of 14 days the moss protonemata were disintegrated via Ultra Turrax Homogenizer. This suspension was used to inoculate 6-1 culture vessels closed with a roll of tissue paper and ventilated with sterile compressed air. Before isolating the organelles the 6-1 culture vessels were darkened for 3 days to get rid of starch and secondary plant compounds which would reduce the organelle yields.

Isolation of protoplasts

Isolation of nuclei

Isolation of chloroplasts (.4) Solutions. Homogenization medium part

Nuclei and nuclear DNA were isolated according to Watson and Thompson [4] with some modifications. The procedure is given below: (A) Solutions. Extraction buffer: 1 M hexylene glycol, 10 mM MOPS-KOH (pH 7.0), 10 mM MgC12, 8 mM L-cysteine, 5 mM fl-mercaptoethanol (added just before use), 0.1% BSA, 1% PVP-10. Gradient buffer stocks: 60% and 30% Percoll (Pharmacia) in washmedium (WM) consisting of 0.5 M hexylene glycol, 10 mM MOPSKOH (pH 7.0), 10 mM MgCle, 5 mM /3mercaptoethanol (added just before use), 0.5% Triton X-100. Lysis buffer: 200 mM Tris--HC1 (pH 8.0}, 50 mM EDTA, 2% sarkosyl. (B) Method. All steps were carried out at 2°C/wet ice. Ten grams of moss protonema were washed with 50 ml ether for 3 rain followed by the same volume of extraction buffer, and homogenized three times with an Ultra Turrax for 15 s. The homogenate was then filtered through 80 ~m nylon mesh. The filtrate was made up to 0.5% Triton X-100, then had to pass one layer of 40/am and a double layer of 20 /am nylon mesh, and was layered on a step gradient consisting of 8 ml 60% and 5 ml 30% Percoll. After centrifugation at 200 × g for 30 rain the nuclei-containing pellet was resuspended in WM and layered on a 5-ml 60% Percoll cushion following centrifugation at 200 × g for 30 min. The pellet was washed with WM and centrifuged at 200 × g for 30 min. The nuclei-containing pellet was resuspended in lysis buffer and, after adding 1 mg/ml Proteinase K, incubated at 37 °C for I h.

For the isolation of chloroplasts and mitochondria protoplasts of Physcomitrella patens were prepared in the following manner [17]: a solution of 0.25% Driselase (Sigma) in redistilled water was centrifuged at 16 000 × g at 4 °C for 20 rain to get rid of the starch. After adjusting the supernatant with 0.45 M sorbitol the protonemata were digested overnight with gentle agitation in the dark at 20 °C. Best yeilds of protoplasts were obtained with 10--12-dayold protonemata.

A (HA): 0.9 M sorbitol, 8 mM EDTA, 1 mM KH~PO 4, 2 mM MgC12, 2 mM MnCle, 20 mM NaC1, 88 mM MES-KOH (pH 5.8). Homogenization medium part B (HB, freshly prepared just before use): 4 mM sodium-ascorbate, 4 mM Lcysteine, 0.2% PVP-10, 6 mM dithioerythritol, 12 mM EDTA, 0.1% BSA. Wash medium: half concentrated HA supplemented with 0.1% BSA. Percoll solution: 50% Percoll, 50% HA (v/v), 1% BSA (w/v). (B) Method. All steps were carried out at 2 °C/wet ice. Protoplasts were separated from undigested material by filtration through a 80 ~m nylon mesh and centrifuged at 50 × g for 15 min. After resuspending in homogenizing medium (HA and HB mixed in a ratio 1 : 1) the protoplasts were homogenized twice for 2 s with an Ultra Turrax. The residue of filtration was again homogenized for 10 s. The combined homogenates were squeezed through a 80 ~m nylon mesh, filtered through four layers of 20 /am nylon mesh and centrifuged at 1500 x g for 3 min. The supernatant was centrifuged again at 1500 × g for 5 min. Pellets were resuspended in wash medium, collected at 1500 × g for 10 min (swing-out-rotor, half strength brake), resuspended in wash medium, and subsequently layered on 50% Percoll. After centrifugation at 115000 × g for 40 rain the chloroplast bands were removed, diluted with wash medium and subsequently filtered through a 10/am nylon mesh. The chloroplasts were centrifuged at 1500 x g for 10 min, resuspeuded in 400 ~ TE-buffer [10 mM Tris--HCl

237 (pH 8.0), 1 mM EDTA] and treated with 40 ~g Proteinase K (30 min, w e t ice). After addition of 40 ~l 0.5 M EDTA (pH 8.0) and incubation at room temperature for 10 min 50 ~l 10% sarkosyl were added, the suspension was gently mixed and incubated for 30 min at 37 °C.

Combined isolation of mitochondria and chloroplasts Mitochondria, chloroplasts, mtDNA and ptDNA were isolated in a combined method according to Albaum [18] with modifications. The protocol of the method is given below: (A) Solution. Homogenization medium part A {HA): 0.8 M sorbitol, 100 mM Tris--HC1 (pH 8.0), 4 mM EDTA. Homogenization medium part B (HB, freshly prepared just before use): 0.4% BSA, 0.2% L-cysteine, 2% insoluble PVP. Wash medium (WM): half concentrated HA. Wash medium/MgC12: WM supplemented with 10 mM MgC12. Wash medium/EDTA: WM supplemented with 50 mM EDTA. Lysis buffer: 50 mM Tris--HC1 (pH 7.5), 15 mM EDTA, 2% SDS. Gradient buffer stocks: 30% and 60% Percoll in WM. (B) Method. All steps were carried out at 2°C/wet ice. Protoplasts were separated from undigested protonemata by filtration through a 80 ~m nylon mesh and centrifuged at 50 x g for 15 rain. After resuspending in homogenization medium (HA and HB mixed in a ratio of 1 : 1) the protoplasts were homogenized twice for 2 s with an Ultra Turrax. The residue of filtration was homogenized for 10 s with the Ultra Turrax. The combined homogenates were squeezed through 80 gin, 40 ~m and a double layer 20 gm nylon mesh. After centrifuging the filtrate at 2500 x g for 3 min the supernatant was centrifuged again under the same conditions, and the pellets were resuspended in wash medium to isolate the chloroplasts. The supernatant was centrifuged at 16 000 x g for 20 min. The mitochondria containing pellet was resuspended in wash mediumfMgC12, incubated with 100 gg/ml DNase I for 1 h at 0°C, filled up with wash medium/EDTA and centrifuged three times at 2500 × g for 3 rain. After centrifugation of the supernatant at 16000 × g for 20 rain the mitochondria were resuspended in lysis buffer

and incubated with 200 ~g/ml Proteinase K at 37 °C for I h. During the DNase I treatment of the mitochondria the resuspended chloroplasts were layered on a 30%/60% Percoll step gradient and centrifuged at 16 000 x g for 30 min. After removing and diluting the chloroplast bands in wash medium they were centrifuged at 2500 x g for 10 min, resuspended in lysis buffer containing 200 ~g/ml Proteinase K and incubated at 37 °C for I h.

Cs C1 density gradient centrifugation The crude organelle lysates were subjected to a CsC1 density gradient centrifugation (initial density: 1.55 g/ml, Beckman VTi65 rotor, 220000 X g, 12 h) in presence of ethidium bromide (1.0 mg/ml) so that the appearing bands could be drawn off under UV-light directly. The DNA was dialysed, precipitated and dissolved in TE-buffer as described in Ref. 19. Bacterial s trains, media and plasmid isolation Bacterial strains and their relevant characteristics are listed in Table I. E. coli strains containing recombinant plasmids were grown on TBY-medium [19] supplemented with 50 ~g/ ml ampicillin. Plasmid DNA was isolated according to Birnboim and Doly [20] with modifications according to Ref. 19. This DNA was subjected to a CsC1 density gradient centrifugation as described above. The supercoiled plasmid DNA was withdrawn, dialysed and precipitated as described [19]. Restriction analysis and gel electrophoresis Organelle and plasmid DNA was digested with the appropriate restriction endonuclease according to the supplier's specifications. Gel electrophoresis was performed in 0.8--1.0% agarose as described [19]. Southern blots and hybridization Alkaline Southern blotting onto GeneScreen plus membrane (NEN/DuPont) was carried out according to Southern [21] and supplier's specificiations. The plasmid DNA fragments were separated

238

on agarose gels, eluted by using a BioTrap (Schleicher and Schuell) and then labelled with [a-8~P]CTP according to the supplier's specifications (BRL nick translation kit). For hybridization the filters were incubated at 42 °C for at least 3 h in presoak consisting of 0.2 g glycin, 3.2 ml double distilled water, 5.0 ml 20 x SSPE [20 x SSPE: 20 mM EDTA, 200 mM NaH~PO~, 3.6 M NaC1, 210 mM NaOH (pH 7.0)], 2 mg salmon sperm DNA, 1.0 ml 100 x Denhardt's solution (100 x Denhardt: 2% BSA, 2% Ficoll, 2% PVP), 10 ml formamide and 60 mg SDS per 20 ml [19]. Hybridizations were carried out in hybridization solution (i.e. presoak without glycine and supplemented with 2.0 g dextrane sulphate and the nick translated probe) in a sealed polyethylene bag for 16--18 h at 42°C. After washing the filters twice in 2 x SSPE, 0.3°/0 SDS and twice in 0.1 × SSPE, 0.3% SDS for at least 15 rain each step at 42°C they were exposed to Fuji-RX film at - 70 °C.

Staining with fluorescent dyes The quality of organelles and the completeness of lysis was controlled by interference contrast microscopy and UV-microscopy. Nuclei and chloroplasts were stained with 2.5 ~g/ml DAPI (4',6-diamidino-2-phenylindole, Serva) and mitochondria were stained with 2.5 ~g/ml DiOC 6 (3,3'-dihexyloxacarbocyanine iodide, Serva) [22]. Electron microscopy Isolated chloroplasts were fixed in 2.5°]0 glutaraldehyde and in 1% osmium tetroxide/ cacodylate buffer [23], dehydrated with acetone, embedded in Spurr medium [24], contrasted with uranyl acetate and lead citrate [25] and examined under a Philips 201 electron microscope. Results and discussion

Large scale culture of moss protonemata As light quality influences the growth of protonemata, we found out that best yields were obtained with a combination of White Light/Grolux Light lamps (Osram). The corn-

pressed air used for ventilation had to be passed through a filter with activated charcoal, otherwise protonema production decreases. We obtained approximately 10 g freshweight of moss protonema per 6-1 culture vessel. In the case of moss protonema this is a fairly good yield, but in comparison with tissue available from higher plants the amount is small. Therefore we had to optimize the isolation methods to get a maximum of organelles and their DNA.

Isolation of nuclei and nuclear D N A The isolation of nuclei and nuclear DNA from mosses is described for the first time. The isolation of moss nuclei according to a former method [4] yielded but a small amount of nuclei. We found that especially the conditions of centrifugation had a major influence on the yield of pure and unbroken nuclei. The protocol given above turned out to be the optimum under our conditions. After incubation of the protonemata with Driselase we obtained only small amounts of nuclei which were contaminated with chloroplasts. Washing the moss protonemata with ether seems to be necessary to get rid of secondary plant compounds which are found in mosses in a remarkable number and quantity [26]. As an average yield we obtained approximately 1.25 x 106 nuclei per gram freshweight. They were obviously unbroken, and the nucleoli were clearly discernible. Microscopical inspection furthermore showed that the fraction was free of chloroplasts or mitochondria. After lysis the nucDNA was purified by CsC1 density gradient centrifugation and digested with the restriction endonucleases Eco RI and Hind III. In moderately loaded gels (2 pg) analysis revealed a restriction pattern of 13 bands (Eco RI) (Fig. 1, lane Ia). When a larger amount of DNA (10 ~g) was loaded, the restriction pattern revealed only five bands and an intense background (Fig. 1, lane IIa). From this we concluded that these five bands represent highly repetitive nucDNA while the other bands seem to consist of moderately repetitive sequences.

239

II S

a

S

III a

S

IV a

W

O

Fig. 1. Restriction and hybridization pattern of nucDNA from Physcomitrella patens. Lane I: Eco RI restriction pattern (2 ~g DNA). Lane II: Eco RI restriction p a t t e r n (10 ~g DNA). Lane III: Hind III restriction pattern. Lane IV: Hybridization of the 82p-labelled pRZ7D insert to the Eco RI digested nucDNA of lane II. s: Molecular weight marker lambda DNA digested with Hind III.

240 Table I.

Gene probes and their relevant characteristics.

Name

Characteristics

Insert

Reference

pCoxIII

Cytochrome-e-oxidase subunit III of Oenothera berteriana 5.8 S, 25 S and 18 S rDNA of Cucurbita pepo 32kd protein of photosystem-IIreaction center of Spinacia oleracea

1.1 kb Eco RI/Pst I subclone in pUC 9 10.1 kb Hind III fragment in pBR 322 769 bp Pst I]Xba I fragment in pUC 18

Hiesel et al. [31]

pRZTD pPSII32/1

In hybridization experiments employing the repetitive 5.8 S, 25 S and 18 S rDNA containing clone pRZ7D (Table I) we observed four distinct signals corresponding to the fragments Eco RI6 (5800 bp), Eco RI-9 (3400 bp), Eco RI-13 (1900 bp) and an Eco RI fragment smaller than 1000 bp {Fig. 1, lane IV). Digestion with Hind III generated a smear and a restriction pattern of 12 clearly visible bands (Fig. 1, lane IIIa). Hybridization with the pRZ7D clone revealed one distinct signal with the fragment Hind III-1 (12 100 bp, data not shown). Hence one can assume that the restriction pattern of Physcomitrella patens nucDNA at least partially consists of repetitive sequences.

Ganal and Hemleben [32] Herrmann (pers. commun.)

i!

Isolation of chloroplasts and chloroplast DNA Driselase digestion of protonemata prior to chloroplast isolation gave higher yields of intact chloroplasts as judged by fluorescent microscopy. Purification by Percoll gradient centrifugation generated two bands of chloroplasts designed as band A and band B. Chloroplasts of these bands were analysed separately by electron microscopy. Both bands contained pure and single chloroplasts with no visible contaminations. Chloroplasts from the lower band B were intact with visible double membranes (Fig. 2b) whereas band A chloroplasts were partially defective (Fig. 2a). To examine the intactness and purity of ptDNA from these two chloroplast bands both were lysed separately. Digestion with the restriction endonuclease Pst I in both cases generated the same restriction pattern of 13 distinct bands larger than 1 kb

Fig. 2. Isolated chloroplasts of Physcomitrella patens analysed by electron microscopy. (a) Chloroplast from Percoll gradient band A with partially defective double membrane. (b) Chloroplast from Percoll gradient band B with intact envelope. Bar represents 1 ~m.

241

I abcd

5

II obcd

111'

S

Sa

Sa

t

Fig. 3. Restriction and hybridization pattern of Physcomitrellapatens ptDNA. Lane I: Pst I restriction pattern of ptDNA from Percoll gradient band A (a} and band B (b) chloroplasts. Hae III restriction pattern of pt DNA (c). Undigested ptDNA (d). Molecular weight marker lambda DNA digested with Pvu II (S). Lane II: Hybridization of the 8~P-labelled pPSII32/1 insert to Pst I (a,b), Hae III (c) and undigested (d) ptDNA. Lane III: Restriction pattern of Sac I/Cla I double digested ptDNA (a}. Molecular weight marker lambda DNA digested with Hind III and Eco RI (S). Lane IV: Hybridization of the 32P-labelled pPSII32/1 insert to Sac I/Cla I double digested ptDNA.

with no visible background for the ptDNAs isolated from band A and band B chloroplasts respectively {Fig. 3, lane Ia,b). From this we inferred that ptDNA could be isolated from both fractions with the same high purity and intactness, although, as shown in the electron microscope, band A chloroplasts had a defective envelope (Fig. 2). Both plastid fractions were pooled. After CsC1 gradient centrifugation, dialysis and precipitation the average yield was 4 ~g

ptDNA per 10 g fresh weight. Calculated from the total DNA extracted and from dot blot analysis we recovered approximately 10% of the ptDNA of the cell {data not shown}. Phenol/chloroform extraction of the chloroplast lysate, extensive Proteinase K digestion, and double precipitation produced higher yields of ptDNA, but this DNA was not pure enough to be digested by several restriction endonucleases. Digestion of ptDNA of the pooled band A

242

II S

a

b

a

III b

a

b

iJ

J

' "41'

~4

i,

Fig. 4. Restriction and hybridization of Physcomitrella patens mtDNA and ptDNA (combined method). Lane h Hae HI restriction pattern of mtDNA [a) and ptDNA (b). Molecular weight marker lambda DNA digested with Hind III (s). Lane II: Hybridization of the ~P-labelled pCoxIII insert to Hae III digested mtDNA (a) and ptDNA (b). Lane IIh Hybridization of the ~zP-labelled pPSII32/1 insert to Hae III digested mtDNA (a) and ptDNA (b).

243 and B chloroplasts with the restriction endonuclease Hse III revealed 30 bands larger than

1 kb (Fig. 3, lane Ic). The double digestion of ptDNA from our

Physcomitrella patens strain with Sac I and Cla I generated a restriction fragment pattern as it has already been published [6] (Fig. 3, lane IIIa). Hybridization with the pPSII32/1 clone (Table 1) showed strong hybridization signals with the fragments P s t I-3 (18 kb, Fig. 3, lanes IIa,b), Hae III-3 (7.5 kb) and a Hae III fragment of approximately 500 bp (Fig. 3, lane IIc). Chloroplast DNA isolated with the combined method could be digested with restriction endonucleases (Fig. 4, lane Ib). This method allows the simultaneous isolation of ptDNA and mtDNA from the same tissue. By this method we obtained a satisfactory restriction pattern of ptDNA in a short time.

Isolation of mitochondria and mitochondrial DNA The isolation of mitochondria and mtDNA from mosses is described for the first time. As in the isolation procedure for chloroplasts Driselase digestion of the protonemata yielded higher amounts of mitochondria and mtDNA. Microscopical control of the isolated mitochondria revealed no contamination by chloroplasts or nuclei. The average yield of mtDNA was estimated to be 1 ~g per 10 g fresh weight. The mtDNA was digested with the restriction endonuclease Hae III generating a restriction fragment pattern of 27 bands larger than 1 kb (Fig. 4, lane Ia), clearly differing from the restriction fragment pattern of ptDNA digested with the same restriction endonuclease (Fig. 4, lane Ib). Hybridization with the mitochondria specific pCoxIII clone (Table I) showed a strong signal with the fragment Hae III-18 (1600 bp, Fig. 4, lane IIa).

Purity of organelle DNA preparations For further testing the purity of the isolated organelle DNA we used clones from higher plants which are specific for nuclear DNA (pRZ7D), chloroplast DNA (pPSII32/1) and mito-

chondrial DNA (pCoxIII) (Table I). Hybridization of nucDNA, ptDNA or mtDNA with the corresponding specific clone resulted in strong hybridization signals after an exposition for 12-- 24 h. When the purity of nucDNA or mtDNA preparations was tested with the non-corresponding clones no hybridization signals could be detected after an exposition for 8 days, whereas ptDNA showed faint signals when hybridized to pRZ7D and no signals when hybridized to pCoxIII. From this we concluded that organelle DNA preparations were highly pure. Furthermore it was important for our work in progress to ascertain the organelle specific character of these clones isolated from higher plants, because there is an increasing evidence for sequence homologies between the three organelle DNA's in several plant species [27-30] while there are no such investigations in lower plants until now.

Acknowledgements This work was supported by a grant of the Deutsche Forschungsgemeinschaft (Ab 10/8-2). We are grateful to Prof. Dr. R.G. Herrmann, Prof. Dr. V. Hemleben and Prof. Dr. A. Brennicke for placing the clones pPSII32/1, pRZ7D and pCoxIII at our disposal. We thank Prof. Dr. M. Mix and E. Manshard for introducing us to electron microscopical techniques. Acknowledgements are also due to Prof. Dr. H.-P. Miihlbach, Dr. W. Kasprik, M. Albaum and K. Hopperton for helpful discussion and critical reading of the manuscript. We wish to thank C. Adami for preparing the photographic work.

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