Mol Gen Genet (1985) 200:503-505 MGG © Springer-Verlag 1985

Short communication

Remarkable restriction pattern differences between green and white branches of variegated 'plastome mutator' plants of Oenothera hookeri

Monika M. Lindenhahn, Michael Metzlaff, and Rudolf Hagemann Department of Genetics, Section Biosciences, Martin-Luther-University, Domplatz 1, DDR-4020 Halle German Democratic Republic

Summary. Green-white variegated plants of the pm line of Oenothera hookeri were reported in the literature to be due to the action of a nuclear recessive 'plastome mutator' (pro). The plastid DNAs of green and white leaves have been studied by restriction analysis and Southern hybridization. Remarkable differences in restriction pattern have been found between them. The green plastids show the typical pattern of plastome I; the white plastids give the pattern of plastome III. The conclusion from our experiments is: variegated 'pm' plants contain two genetically different types of plastids. Their variegation is not due to nuclear gene-induced plastome mutations, but is the result of hybrid bleaching of the type III plastids, which were combined with type I plastids following - as we suppose - inadvertent pollination.

The mutability of plastid genes, of "the plastome", has been the subject of numerous investigations. Nevertheless many questions remain. We have approached this field in two ways:

(1) We have used chemical mutagens to induce plastome mutations in higher plants. Antirrhinum majus turned out to be a particularly suitable subject for the induction of plastome mutations by nitroso-methyl-urea (NMU) and ni- troso-ethyl-urea (NEU); Oenothera hookeri and Lycopersi- con esculentum could be successfully treated too (Hagemann 1976, 1979, 1981; Hagemann and Lindenhahn 1983; Hos- ticka and Hanson 1984).

(2) Nuclear gene-induced plastome mutations have been reported for several plant species. We especially studied the 'albostrians' line of Hordeum vulgare (Hagemann and Scholz 1962; Hagemann 1979). Epp (1973) described a var- iegated line of Oenothera hookeri (strain Johansen), whose variegation he attributed to a nuclear 'plastome mutator' (pm) gene. At present we have two types of variegated Oenothera plants available in our laboratory; (a) variega- tions induced by NMU treatment and (b) variegated plants of Epp's pm line. To compare these two types of variegated plants, we have performed - among other studies restric- tion analyses of the plastid DNAs of the green and white (or yellow) plastids.

The aim of our experiments was to compare the plastids of the green and the white branches of individual plants

Offprints requests to: M.M. Lindenhahn

separately. To do this we used a technique described by Atchison et al. (1976), which needs only very small amounts of plant material and was particularly suitable for our inves- tigations. Studies on plastid DNAs of Pelargonium and Beta confirmed the suitability of this method (Metzlaff et al. 1981, 1982). Using the Atchison-technique we determined the restriction patterns of the plastid DNAs of Oenothera plants after double digestion with KpnI and SalI as well as with PstI and SalI (Additional digests were performed with EcoRI and BamHI respectively). Gordon et al. (1982) analysed the restriction patterns of the plastid DNA of the five wild-type plastomes I, II, III, IV and V of the subsec- tion Euoenothera; we used their findings as a reference.

Restriction analyses of the DNA of green and white plastids of variegated plants of the 'plastome mutator' (pro) line revealed very unexpected results (Fig. 1), which may lead to quite another interpretation of the whole phenome- non:

(1) Plastid DNA from green control plants of Oenothera hookeri exhibits the restriction pattern described by Gordon et al. (1982) for plastome I of Oenothera, e.g. Oenothera hookeri.

(2) Green branches of variegated pm plants have plastids the DNA of which shows the same type I restriction pattern as control Oenothera hookeri plants, i.e. they contain nor- mal hookeri plastids. In many parallel experiments the plas- tid DNA from green Oenothera hookeri plants (control) as well as from green leaves of variegated pm plants exhib- ited exactly the same restriction pattern of plastome I, after KpnI/SaII and PstI/SalI double digestions (Fig. 1) and also after digestions with EcoRI or BamHI.

(3) However, the plastid DNA of white branches of variegated pm plants shows a restriction pattern, which is remarkably different from type I (Oenothera hookeri), but is identical with the restriction pattern of plastome type III of Oenothera (normally found in Oenothera lamarckiana), as described by Gordon et al. (1982), for PstI/SaII, Kpnl/ SaII, EcoRI and BamHI digestions.

(4) The fact that the green branches of variegated pm plants contain type I plastids, whereas the white branches contain type III plastids was additionally demonstrated by Southern hybridizations (Fig. 1). In contrast to the studies of Gordon et al. (1982) instead of purified spinach chloro- plast 16S rRNA we used a gene probe containing the total 16S rDNA of spinach and additional DNA sequences up- stream and downstream of the gene. In all our hybridization patterns we can detect the same bands as Gordon et al.

504

Md 20

1 0 ~

7 ~

5 ~

3 m

a b c d e f g h i k I

m

m

I'

Fig. 1. Comparison of restriction patterns of ptDNAs of green (type I : a, c, i, 1) and white (type III: b, d, k, m) leaves of Oenothera hookeri, line 'pro'. Lanes a-d show the 0.6% Seakem agarose restriction patterns resulting from double digestions with PstI and SalI, lanes i-m show double digestions with KpnI and SalI. Lanes e-h show the patterns of hybridization to the corresponding restriction patterns using a 16S rDNA probe of spinach; e and g contain ptDNA of green leaves, f and h ptDNA of white leaves (all tracks shown in this Figure were run in the same gel)

(1982), and some additional bands due to the bigger size of the probe used. (This gene probe was kindly provided by Professor R. Mache, University of Grenoble, France.)

The hybridization patterns are typical for type I plastids (green branches) and for type III plastids (white branches).

(5) Sears (1983) mentioned a change in the restriction pattern of variegated pm plants and discussed an interpreta- tion assuming the presence of transposable elements. Cer- tainly, clear results can be obtained only when individual green and white branches of defined variegated plants are studied. The Atchison-technique, which allows the produc- tion of clear restriction patterns from very small leaf sam- ples, is a prerequisite for the elucidation of these differences.

The results reported lead us to doubt the interpretation, proposed by Epp (1973) and accepted in many review arti- cles and monographs (e.g. Kirk and Tilney-Bassett 1967; Herrmann etal. 1980; Sears 1983). Our investigations, which are continuing, lead us to the following hypothesis:

(a) The green-white variegated line of Oenothera, origi- nally studied by Epp (1973) and now kept in several labora- tories, is not characterized by frequent gene-induced plastid mutations. It rather represents an example o f ' hybrid varie- gation' , well known in Oenothera since the classical studies of Renner (1922, 1936), but also found in other genera, e.g. Pelargonium (Review: Hagemann 1964; Metzlaff et al. 1982).

(b) Epp (1973) described that the first variegated plant (E-15-7) of this line originated in an M z after treatment of Oenothera hookeri seeds with ethyl-methanesulphonate (EMS). It is known, that EMS often induces severe sterility in the treated plants. We therefore suppose that the partially sterile M~ parent was unintentionally cross-pollinated by another Oenothera plant containing type I I I plastids.

(c) The studies of Stubbe (1959, 1960, 1964) and Sch6tz (1958) have demonstrated, that a combination of the geno- me AA, e.g. from Oenothera hookeri, with plastome III leads to hybrid bleached type III plastids; [compare the "combinat ion chart" , (Stubbe 1959, 1960, 1964)]. Our find- ing, that the type III plastids are white (or yellowish) in combination with the hookeri genome, is thus in full accor- ance with these facts.

Further studies are under way in our department on the different types of green-white variegations of Oenothera.

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Communicated by H. Saedler

Received March 18/May 29, 1985