Detection of the proplastid ribosomal RNA genes in rice embryos

Plant Science Letter~, 18 (1980) 13--17

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© Elsevier/North-HollandScientific Publi~he~ Ltd.

DET~ON OF THE PROPLASTID RIBOSOMAL RNA GENES IN RICI~ EMBRYOS

MASAHIRO SUGIURAand KIYOHARUOONO* National Institute of Genetics. Mi~hima, 8hizuoka-ksn 411 and *National Institute of Agricultural $cience~, T~ukuba, Ibar~i.ken 305 (Japan)

(Received November 8th, 1979) (Reviaion received December 10th, 1979) (Accepted Deeembe~24th, 1979)

SUMMARY A DNA fragment which contains rice proplastid rRNA gene~ was detected after EcoRI digestion of rice embryo DNA followed by Southern image hybridisation. The 7.5 ×. 106 mol. wt fragment codes for 2~S, 168 and 58 • rRNAs. INTRODUCTION Plastids of higher plant~ contain 70S ribosomes, and these ribosom~ contain 23S, 16S, 5S and 4.5S rRNAs [1]. Plastid DNAs e x i t as circul~ molecules with molecular weights of approx. 108 and code for.all pl~tid rRNA species [2]. The structure of chloroplast rRNA gene~ from plant leaves have extensively studied in maize [3], spinach [4,5] and tobacco (ref. 6 and Kusuda et aL, unpublished). However, little information i~ available on the rRNA genes in proplastids or leucoplasta Recently, we detected the rice proplastid rRNA gene~ in the DNA preparation from dried rice seeds without purification of the proplastid DNA. MATERIALS AND METHODS Rice e m b r y o D N A . Rice seeds (Oryza sativa L. vat. Manget~umochi) were obtained from the Experimental field of the National Imtitute of Agricultural Sciencea Rice embryos were prepared as described previously [7]. Rice embryo DNA was extracted from the fraction enriched in nuclei of rice embryos as described [7], except that the final purification step for nuclei was omitted. r R N A s . Rice cytoplasmic 25S and 17S rRNAs were prepared as described [7]. Tobacco chloroplast 23S, 16S and 5S rRNAs were extracted and labelled

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Tobacco chloroplast 23S, 16S and 5S rRNAs were extracted and labelled in vitro with 32p using T4 polynucleotide kinase [8] as previously reported

[6]. Southern image hybridisation. Ten micrograms of rice DNA were digested with 10 units of EcoRI {Bethesda Research Labs) in a 100 ~l reaction mixture containing 100 mM Tris--HC1 (pH 7.5), 50 mM NaCI, 10 mM MgC12, 0.1 mM EDTA for 5 h at 37°C. The digested DNA was ethanol precipitated, taken up in 30 ~l solution containing 8% sucrose and 0.02% bromophenol blue and applied to a 0.7% agarose (Sigma, Type II) slab gel (an 8 mm × 5 mm well, 11 cm height) in 40 mM Tris, 20 mM sodium acetate, 2 mM EDTA, acetic acid (pH 7.8) and 0.5 ~g/ml ethidium bromide. Gels were run at 20 V for 15 h. The DNA fragments were denatured, transferred onto nitrocellulose filter strips (Schleicher & Schiill, BA85) and

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Fig. 1. Agaro~e gel electrophoresis o f Hind III fragments o f ~ DNA c o n ~ i n l n g a Sst I fragment o f pTCP6 DNA ( t o o l w~ 8.7 × 106) as size marker (A) and EcoRI fragments o f rice e m b r y o DNA (B). Autoradiog~ams o f Southern images o f EcoRI digested rice e m b r y o DNA hybridised to plastid 3~P-labe|led 23S + 16S rRNAs in the absence (C) or presence (D) of excess unlabelled cytoplasmic 25S and 17S rRNAs as competitors. Numerals indicate molecular weights (× 10-6 ).

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Fig. 2. Autoradiograms o f Southern images of EcoRI digested rice e m b r y o DNA hybridi~ed to pla~tid 32P-labelled 23S, 16S or 5S rRNA. The 32P-labelled 238, 168 and 58 rRNA probes contained unlabelled 16S + 5S, 23S + 5S and 23S + 16S rRNAs, respectively. All the probes contained unlabelled rice cytoplMmic 25S + 17S rRNA~. Numera~ indicate molecular weights (× 10-6).

hybridised with the 32P-labelled rRNAs as described b y Southezn [9] with modifications [6]. RESULTS

AND

DISCUSSION

Rice DNA was extracted from the fraction enriched in nuclei o f rice embryos, because it was rather difficult to prepare high molecular weight DNA from total rice embryos. This fraction still contained portions of the proplastids. The DNA was completely digested with EcoRI and fractionated by 0.7% agarose gel electrophomsis. As shown in Fig. 1B, two discrete bands with molecular weights o f 5.2 X 10 6 and 5.0 × 10 6 could be identified in an EcoRI digest o f the rice DNA. These two bands have been shown to contain rice cytoplasmic rRNA genes [ 7]. The DNA fragments in the gels were denatured and t r a n s f e n ~ l t o nitrocellulose filter strips. Tobacco chloroplast rRNAs labelled with 32p were used as R N A probes, because the competition hybridisation and the thezmal

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stability experiments have shown that the nucleotide sequences of chloroplast rRNAs were very similar in higher plants [10]. A mixture of the chloroplast 23S and 16S rRNAs was hybridised to filter-bound rice DNA fragments. The resulting autoradiogram as in Fig. 1C showed that the rRNAs hybridised to three distinct fragments. Using ), Hind III fragments and a pTCP6 Sst I fragment (rnoL wt 8.7 × 106 [Kusuda et al., unpublished]) as size markers, the size of t h e hybridised fragments were estimated to be of tool. w t 7.5 × 106, 5.2 × 106 and 5.0 × 106 . When hybridisation of the chloroplast 3~P-labelled rRNAs was carried o u t in the presence of a b o u t 100-fold each of unlabelled rice cytoplasmic 25S and 17S rltNAs as competitors, the tool. wt 5.2 × 106 and 5.0 × 106 bands in an autoradiogram almost disappeared. The presence o f c o m p e t i t o r R N A was necessary to ensure specificity of hybridisation. These results indicate that rice proplastid rRNA genes are located on the tool. wt 7.5 × 106 fragment of the E c o R I digest. Although t o b a c c o chloroplast rRNAs hybridised in some extent to rice cytoplasmic rDNA, rice cytoplasmic rl~NAs did not hybridise to t o b a c c o chloroplast DNA (data n o t shown). It is unlikely that the fragment was originated from the mitochondiral DNA because the mitochondria were a minor contaminant in the fraction enriched in nuclei due to their small size [11] and the contents of mitochondrial DNAs were much less than that of plastid DNAs [12]. In order to ensure the location of the proplastid 23S, 16S and 5S rl~NA sequences on the tool. wt 7.5 × 106 EcoRI fragment, the Southern images of rice DNA digested with Ecol~I were hybridised separately with 32P-labelled 23S, 16S and 5S rl%NAs in the presence of approx. 100-fold each of unlabelled c o m p e t i t o r rltNAs. Figure 2 shows that the 23S, 16S and 5S rRNAs hybridised to the tool. wt 7.5 × 106 fragment. This fragment has enough size to contain a set o f chloroplast 23S, 16S and 5S r R N A sequences 0 I

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~ 16S 23S 5S ~ D Fig. 3. Diagrammatic representation of the organkation of plastid rRNA genes. A, Tobacco [14]; B, Spinach [4]; C, Maize [3]; D, Rice. A scale indicates molecular weight~ (× 10 -~) and arrows EcoRI sites.

17 and t h e spacer regions. The EcoRI cleavage map of the rRNA gene region of rice plastid DNA is different from t h a t o f tobacco or spinach chloroplast DNA [4,5,14], b u t it seems to resemble t h a t of maize chloroplast DNA [3] (Fig. 3). At present it is quite difficult to isolate proplastids in large _~mounts for biochemical analyses. This procedure enables us to detect proplastid rRNA genes in total cell DNA. This technique requires labelled RNA probes with high specific activity. These RNA probes can be easily obtained by labelling RNA in vitro with 32p using T4 polynucleotide kinase and [7-32P]ATP [8,13]. The procedure reported here may be useful to compare the organisation o f plastid rRNA genes in m a n y different plant species. ACKNOWLEDGEMENTS We t h a n k Miss Y. Tsuchiya and Miss M. Suzuki for excellent technical assistance and Dr's. J. Kusuda and F. Takaiwa for preparation of tobacco chloroplast rRNAs. REFERENCES

1 P.R. Whitfeld, Chloropkst RNA, in P.R. Stewart, D.8. Letham (Eds.), The ribonucleic acids, 2nd ed., Springer, Berlin, 1977, p. 29"/. 2 J.R. Bedbrook and R. Kolodner, Annu. Rev. Plant Physiol., 30 (1979) 593. 3 J.R. Bedbrook, R. Kolodner and L. Bogorad, Ceil 11 (1977) 739. 4 P.R. Whitfeld, R.G. Herrmann and W. Bottomley, Nucleic Acids Res., 5 (1978) 1741. 5 H.J. Bohnert, K. Gordon, A.J. Driesel, E.J. Crou~ and R.G. Herrmann, in Akoyunoglou et al. (Eds.), Chloroplast development, North-Holland, Amsterdam, 1978, p. 569. 6 M. Sugiura and J. Kusuda, Mol. Gen. Genet., 172 (1979) 137. 7 K. Oono and M. Sugiura, Chromosoma, in pres~. 8 M. Sugiura and M. Takanami, Proc.. Natl. Acad. ScL U.S.A., 58 (1967) 1595. 9 E~M.Southern, J. MoL BioL, 98 (1975) 503. 10 J.R. Thomas and K.K. Tewari, Pro©. Natl. Acad. Sci. U.S.A., 71 (1974) 3147. 11 K. Ueda smi H. Tsuji, Protopksma, 73 (1971) 203. 12 M. Ishids, Nuclear and organelle DNAs, in H. Kihara and K. Hayashi (Eds.), Plant genetics, Vol. 2. Sh6kabo, Tokyo, 1977, p. 1 (in Japanese). 13 N. Maizels, Cell, 9 (1976) 431. 14 F. Tilr~iwa and M. Sugiura, Gene, in press.