Plant Molecular Biology Reporter pages 104-113 Volume 8(2) 1990

Protocol

Preparation of Mitochondrial DNAs from Sunflowers (He l ian thus annuus L.) and from Beets (Beta vu lgar i s ) Using a Medium with a

High Ionic Strength

Christine Pdrez, Jean-Francois Bonavent and Andrd Bervilld

Institut National de la Recherche Agronomique, Station de G6n6tique et d'Am61ioration des Plantes, Laboratoire de G6n6tique et Mutagen6se,

BV 1540, 21034 Dijon Cedex, France (A.B.) Telephone: 80-63-31-55, Fax: 80-62-32-63

Key words: DNA, gel electrophoresis, RFLP, mtDNA, cell organelles.

Abstract: The isolation of mitochondria from sugar beet and sunflower has been carried out in a buffered medium of high ionic strength (1 = 1.40 M). The m tDNAs obtained were enriched for supercoiled molecules of low molecular weight. Chloroplast and mitochondrial DNA were successively prepared and used from a single leaf preparation for further analysis of restriction fragment length polymorphism. The few available wild relatives of sugar beet or sunflower have also been analyzed by comparison of the restriction patterns of their chloroplast and mitochondrial DNAs.

A prerequisite for studying the cytoplasmic variability of a crop and its wild relatives is to se tup a convenient method to prepare both their ctDNA and their mtDNA. The method should be

easily adaptable from one ecotype to another and, since the wild relatives are difficult to maintain, the method must be compatible with limited plant material and allow for successive isolations of c tDNA and mtDNA from a single preparation. The methods previously described by Leroy

Abbreviations: CCC, covalently closed circular; CMS, cytoplasmic male sterile; DEPC, diethyl pyrocarbonate;MF, male-fertile; MS, male-sterile; OC, open circle; pO, minicircle c of sugar beet in mitochondria; p1T, mitochondrial plasmid of sunflower.

104

Mitochondrial DNAs from Sunflowers and Beets 105

et al. (1985) for sunflower or by Powling (1982) or Mikami et al. (1984) for sugar beet are unsuitable because of the lack of either a storage root or of sufficient fresh weight of tissue. Furthermore the DNAs obtained from mature leaves through these methods often cannot be hydrolyzed by restriction enzymes. Consequently a fast, comparative s tudy of ecotypes cannot be made.

We present a method that has successfully been used to obtain ctDNA and mtDNA from sunflower and sugar beet. We have found that the main advantage of this method is the level of recovery of the CCC form relative to OC form of a plasmid particularly for the plT of sunflower and the pO of sugar beet.

Solutions Required

Homogenizat ion buffer solution A 1 50 mM Tris, pH 8.0 1.3 M NaCl 25 mM EDTA 56 mM mercaptoethanol 0.2% half-skimmed milk 2

Buffer B same as buffer A except 1.25 M NaC1 0.2% PEG 0.2% half-skimmed milk

Lysis buffe r C 25 mM Tris, pH 8.0 20 mM EDTA 2% SDS 0.5% 2-mercaptoethanol 0.4% DEPC

Procedure

Plant material Beta vulgaris, wild forms of Beta maritima, Helianthus annuus and wild forms of Helianthus petiolaris subspecies fallax were grown in a green

106 Pdrez et al.

house for two to three months. Etiolated sunflower seedlings were grown at 25~ for seven to ten days.

Preparation of mitochondria The procedure was extended from the one of Bookjans et al. (1984) to prepare chloroplasts. For Helianthus species the steps were as following:

Portions of 50 g fresh weight were homogenized in 250 ml of bu ffer A2. 3. Centrifugations were 500 x g for 10 min to pellet debris followed by 2,600 x g for 15 min to sediment chloroplasts. The pellet was dissolved in starting volume, centrifuged twice more, and the supernatant run again at 18,000 x g for 20 min to sediment mito- chondria.

Lysis of mitochondria �9 Mitochondrial pellets were resuspended in 6 ml buffer C 4. �9 After 30 min 4 at 37~ with intermittent agitation, the tubes were

chilled for 20 min on ice.

Protein elimination �9 Cold potassium acetate to 1.2 M final concentration was added,

mixed gently by inversion, and kept on ice for 30 min. �9 The K+-acetate-SDS-protein complexes were removed by centrifu-

gation for 10 min at 12,000 x g and discarded. �9 The supernatants were collected by filtration through nylon cheese-

cloth and collected in an erlenmeyer for further purification by the phenol-chloroform method, and then ethanol-precipitated in the presence of 0.5 M ammonium acetate.

Mitochondrial DNA recovered �9 The nucleic acid pellets were dissolved again in 0.4 ml sterile water

for RNAase s treatment by a mixture of RNAase A (5 mg/ml) and RNAase T1 (100 U/IJI) for 30 min at 37~

�9 After two subsequent chloroform treatments and a final ethanol precipitation, the mtDNA was resuspended in 501Jl buffer TE, pH 7.6.

Probes The pO clones correspond to the minicircle c of sugar beet linearized by EcoRI and cloned in the EcoRI site of the pBR322 vector (Hansen and Marcker, 1984). Southern transfer, preparation of [32p]-labelled probe were done according to Faivre-Rampant et al. (1989). Specific activity of the probe pO was 109 dpm-12g -1 DNA.

Mitochondrial DNAs from Sunflowers and Beets 107

Notes

1. For Beta species 100 g of leaves were ground in 200 ml homogeniza tion buffer B. It appears to be necessary to maintain a high level of antioxidant during all the steps of the organelle preparation to obtain a restrictable DNA prepa- ration. The 2-mercaptoethanol concentration we used is ten times higher than the one used by Kemble et al. (1980), Brown et al. (1986) or Bookjans et al. (1984).

2. Bovine serum albumin replaced by half-skimmed milk. 3. For Beta species 1.25 M NaC1, 0.2% PEG 6000. This concentration in NaC1

appeared less effective for the yield in mtDNA for sunflower. Most of the molecules giving a large fluorescent spot under UV, possibly phenolic com- pounds, were eliminated by PEG 6000 but not by PVP or Polyclar.

4. In the buffer D, DEPC and 2-mercaptoethanol were omitted but 0.07% self- digested pronase (w/v) was added. Sugar beet mitochondria were lysed for I hour at 37~ in 6 ml buffer D. For Helian thus, we observed that the lysis step mus t be done in the presence of DEPC; otherwise the DNA is not restric table. The pronase step prior to potassium acetate-SDS precipitation was omitted for sunflower mtDNA because it does not improve the quality or quantity of mtDNA obtained.

5. We eliminated RNA by treatment with RNAase rather than Bookjans's method because of the small quantity of DNA obtained.

Resul t s

The prepara t ion of m t D N A with the me tho d described was part icu- larly efficient for compar ing the content of p lasmids of Helianthus and Beta species as shown in figures I and 2, respectively. Several plasmid bands of different mobili t ies are visible. For sunf lower carrying normal (male-fertile) cy toplasm (CNP3-MF in Fig. 1), the two bands cor respond to the CCC and the OC of the piT plasmid (P6rez et al., 1988). For the subspecies fallax, several bands are visible (Hpfallax), so we cannot recognize those cor responding to the CCC and OC forms of the same plasmid. These plasmids are not all c loned yet, so that we used other p lasmids to p robe the Southern transfer. One of the Petiolaris fallax plasmid shares homologies with piT f rom sunf lower and pO from sugar beet (data not shown, see Fig. 1 of P6rez et al., 1988). Tw o signals at 2.3 kb and 1.0 kb of apparen t molecular weight cor respond to a 2.1 kb circular molecule. The CMS Leclercq cytoplasm nei ther contains plas- mid (CANP3-MS in Fig. 1) nor displays any signal with ei ther plT and pO as p robe (not shown).

Restriction f ragments of nat ive mtDNAs from several species or lines of Beta were separated by elctrophoresis and stained with e th id ium bro- mide (Fig. 2). Variations in the distr ibution of low molecular weight

108 P&ez et al.

Fig. 1. Plasmid sets of mitochondria pre- pared from Helianthus species with the high ionic strength grind- ing medium. 1-kb lad- der (BRL); CANP3 MS, Helianthus annuus, male-sterile line CANP3; Hpfallax, H. pe- tiolaris fallax ecotype 200; CANP3 MF, H. an- nuus, male-fertile line CANP 3. Twentygrams of leaves was used to obtain profiles.

DNA are revealed. Plasmid forms (num- bered 1 to 4) are vis- ible in 12 samples, making the compari- son between species difficult. Figure 2 with Southern trans-

fer also shows probed pO plasmid labelled with 32p. Clearcut differences are revealed between samples displaying signals due to the CCC, linear, and OC forms, and those without signal. The similarity of the plasmid sets of the genotypes, however, was closely related; hybridization re- veals that the distribution of the pO plasmid (number 2) in the Vulgares section (FB and SB) is not tightly related to the normal or the CMS cytoplasm. The species belonging to sections of Beta, namely Corollinae and Patellares, do not carry plasmids homologous to pO (numbers 3 and 4); thus these plasmids will have to be cloned for further studies.

Mitochondrial DNAs obtained with this method were also hydro- lyzed by restriction enzymes as shown in figure 3.

Mitochondrial DNAs from Sunflowers and Beets 109

Discuss ion

We report here the use of a medium of high ionic strength to isolate mitochondria of sugar beet and sunflower. The method proved useful for the simultaneous preparation of ctDNA and mtDNA from plant material which has to be analyzed in routine or in screening, i.e. wild ecotypes of sugar beet and sunflower especially rich in phenolic com- pounds (P6rez et al., 1988; Bonavent et al., 1989). The principle of the method to obtain chloroplasts was explained by Bookjans et al. (1984) for the preparation of ctDNA from chloroplast-deficient mutants of peas.

Fifty grams fresh weight of sunflower leaves currently yields 101Jg of mtDNA and 50 lag of ctDNA. The yield of chloroplast DNA is half that of Bookjans et al. (1984) for peas. Neither the yield of mtDNA nor of ctDNA has been improved (10 to 15 lag mtDNA per 100 g fresh weight). For mtDNA from sunflower seedlings, however, the yield is about twice that obtained by Leroy et al. (1985).

The volumes of mitochondrial matrix, excluded by sucrose ~K2 but not 3H20, are ten times as high for maize as for sunflower (0.2 lal/g fresh weight) (Bervill6 et al., 1984; P6rez, 1987). If the yield of mitochondria from sunflower is in the same ratio, these results could explain the low quantity of mtDNA obtained from fresh sunflower tissue by the method previously published (Leroy et al., 1985; Brown et al., 1986).

For the beet preparations, the yields (60 to 100 lag per 100 g fresh weight) of ctDNA are higher than for sunflower. We did not compare them to the yields of other methods because the saline method is as suitable for ctDNA as for mtDNA. The main advantage for the sunflower and its relatives is the enrichment of the CCC form of the mitochondrial plasmids. Mitochondrial DNA can also be obtained from etiolated seedlings with a very high yield of CCC molecules of low molecular weight as verified by hybridization experiments (P6rez et al., 1988). The hybridization signal of the band corresponding to the CCC form of the plT plasmid is much more intense than that of the OC form; the linear form is not detectable.

We reduced the steps for the purification of the DNA in order to carry out a simple method to compare ecotypes (often having limited amount of stored material) and to adapt it to various organs or tissues.

More extensive studies are needed to determine the effects of the higher sodium concentration (1.3 M) used in the saline preparation and

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Fig. 3. Restriction endonuclease profiles of mitochondrial DNA prepared with the high ionic strength medium. (left) EcoRI profiles for sunflower lines. 1-kb ladder (BRL); CCM 61 MF, leaves of H. ann u us MF line CCM 61; 2603 MF, leaves of H. annuus, MF line 2603; CVH11 MF, H. annuus, MF line CVH11; CA4 MF seedl., seedlings of H. an n u u s, MF line CA4; CA4 MF lea f, leaves of H. an n u us, MF line CA4. (right) HindI I I profiles for Beta species. Bvu159, MS sugar beet line 59; Bvul CS, table beet Crapaudine Semaphor; Bvul 1030, MS sugar bee t line 1030; Bvu11030, MF sugar beet line 1030.

112 Pdrez et al.

the effects o f the acce le ra t ion a n d d u r a t i o n of cen t r i fuga t ion . D u r i n g o u r e x p e r i m e n t s , w e o b s e r v e d tha t the s e d i m e n t a t i o n o f ch lo rop l a s t s a n d m i t o c h o n d r i a w a s p e r t u r b e d in a m e d i u m of h i g h ionic s t r e n g t h c o m - p a r e d to a m e d i u m c o n t a i n i n g 12% sucrose . C h l o r o p l a s t s b e c a m e l ighter a n d m i t o c h o n d r i a dense r . To a v o i d c o n t a m i n a t i o n of c h l o r o p l a s t s b y m i t o c h o n d r i a , w e s h o r t e n e d the c h l o rop l a s t c e n t r i f u g a t i o n step.

W e h a v e f o u n d this p r o c e d u r e to be su i tab le a l so for l up ine a n d s u n f l o w e r . W e expec t tha t it will be a m e n a b l e to a w i d e va r i e t y of o t h e r plants .

Acknowledgements: We thankJacky Delbut for his help in photographic prints. This work was supported by the GRANT N~ 4439 from INRA and MRT to Andr6 Bervill6.

References

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Mitochondrial DNAs from Sunflowers and Beets 113

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