Chloroplast DNA deletions associated with wheat plants regenerated from pollen: possible basis for maternal inheritance of chloroplasts

Ceil, Vol. 39, 359-368, December 1984 (Part l), Copyright0 1984 by MIT 0092.8674/84/120359-IO $02.00/O

Chloroplast DNA Deletions Associated with Wheat Plants Regenerated from Pollen: Possible Basis for Maternal Inheritance of Chloroplasts

A. Day and T. H. N. Ellis John lnnes Institute Norwich NR4 7UH, England

Summary

Albinism in plants regenerated from pollen by anther culture may result from the same mechanisms as postulated to be responsible for maternal inheritance of chloroplasts. Consistent with this view, Southern blotting was used to show that large regions of the chloroplast genome (over 80% in one case) were deleted in the major ctDNA molecules present in eight albino wheat plants regenerated from separate pollen calluses. A wide variety of deleted ctDNA molecules was present in these plants. Most albino plants appeared to contain a heterogeneous population of ctDNA molecules. Al- bino plant 6 contained one major type of deleted ctDNA molecule that had a circular 39 kb restriction map. The retention of only two Pst I (unaltered in size) fragments in albino plant 7 may delimit a region of ctDNA that contains a replication origin.

Introduction

A doubling of the chromosome complement of haploid plants provides a rapid means of obtaining homozygous diploid plants for use in plant breeding programs (Snape, 1981). Widespread use of this technique in crop plants requires a convenient and reliable procedure for producing large numbers of haploid plants. Anther culture is one method that is widely used to produce haploid plants (Maheshwari et al., 1982). In anther culture, plants are regenerated from immature pollen grains that are formed by the meiotic division of pollen mother cells. Anther culture procedures adopted by different laboratories show a great deal of variation. The basic steps used for cereal anther culture in this institute are illustrated in Figure 1. Anthers dissected from young spikes are subjected to a temperature stress and floated on a liquid medium in which they dehisce, thereby releasing pollen into the medium (Huang and Sunderland, 1982). Some of this pollen divides to form calluses which are transferred to solid regeneration medium to induce the formation of shoots and roots. In the cereals and grasses, plants regenerate solely from pollen callus, whereas in many other species proembryos are formed directly from pollen (Sunderland and Dunwell, 1977). Plant yields are low and, in the case of cereals and grasses, a high percentage (100% in some cases) of the plants regenerated are albino. Albinism in anther culture of cereals and grasses is one of the major factors that has impeded the use of this technique in cereal breeding programs. In wheat anther culture, the percentage of albino plants produced is influenced by the genotype and phys- iological state of the anther-donor plants, in addition to the conditions of the culture process (Bullock et al., 1982;

Ouyang et al., 1983). However, the primary cause of this albinism is not known.

Vaughn and coworkers (1980) have suggested that maternal inheritance of chloroplasts is due to an alteration of this organelle inpollen, and that the same process may be responsible for the generation of albino plants in cereal anther culture. Electron microscopical studies have shown that these plants contain proplastids (Clapham, 1977) which are thought to be precursors of chloroplasts (Whatley, 1977). Rice albino pollen plants lack the 23s and 16s chloroplast ribosomal RNAs and ribulose-I,5 bisphosphate carboxylase (Sun et al., 1979). The large subunit of this enzyme and the chloroplast ribosomal RNAs are known to be encoded by the chloroplast genome (Bedbrook et al., 1979; Bedbrook and Bogorad, 1976). We set out to establish whether the absence of these chloroplast encoded products was associated with a defective chloroplast genome. Genetic analysis was not possible because diploid albino plants remained vegetatille and did not flower. We therefore used a molecular approach. Restriction enzyme digestion patterns of chloroplast DNA (ctDNA) from green and albino tissue were compared. Our results show that large regions of the chloroplast genome were deleted in most wheat albino plants.

Results

ctDNA Is Present in Albino Pollen Plants Wheat ctDNA has been mapped with Pst I (Bowman et al., 1981). In addition, a number of Barn HI fragments have also been mapped (Bowman et al., 1983). Comparisons between ctDNA sequences in green leaves from seed plants and albino leaves from pollen plants were made by probing total DNA extracts digested with Barn HI and Pst I on Southern blots (Southern, 1975). The locations of cloned ctDNA fragments that were used as hybridization probes are indicated on the restriction enzyme map of wheat ctDNA (Bowman et al., 1981; Bowman et al., 1983) shown in Figure 2. All pTac plasmids contained wheat ctDNA restriction fragments and were a gift from C. M. Bowman and T. A. Dyer, Plant Breeding Institute, Cam- bridge. We were also able to use pHvc plasmids that contained restriction fragments of barley ctDNA as hybridization probes because of the conservation of DNA sequence homology and DNA sequence arrangement between the barley and wheat chloroplast genomes (Poulsen, 1983).

DNA sequences homologous to ctDNA have been found in mitochondrial DNA (Stern and Lonsdale, 1982; Lonsdale et al., 1983; Stern and Palmer, 1984) and nuclear DNA (Timmis and Scott, 1983). In tissues where ctDNA levels are low, e.g., root tissue, hybridization of cloned ctDNA fragments to ctDNA sequences present in other organellar DNAs can make the identification of true ctDNA restriction fragments more difficult, particularly in cases where the chloroplast genome is altered. pTac B2 contains a 9.6 kb Barn HI fragment of wheat ctDNA that is known to contain the genes coding for the large subunit of ribulose-1,5-

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C Float Anthers from Spikes on Callus Medium

- Transfer Calluses onto Regeneratron Medium

Figure 1, Basic Steps of Barley Anther Culture (Courtesy of J. M. Dunwell and N. Sunderland)

Tillers are surface-sterilized with ethanol before the spikes are dissected out under aseptic conditions. Spikes are subjected to a temperature stress (14-28 days) before excising the anthers. These are placed on a callus-inducing medium. Calluses larger than 1 mm (formed after 21-40 days) are transferred to a solid regeneration medium. Some of these calluses begin to differentiate into leaves after a minimum of 21 days.

Barn Hl

Figure 2. Pst I Restriction Map of Wheat ctDNA Also Showing the Location of Three Barn HI Fragments

Wheat Pst I and Barn HI ctDNA fragments are represented by a letter identifying the restriction enzyme producing it and a number in order of decreasing size. Sizes of these fragments are given in kb. The positions of restriction fragments used as hybridization probes are indicated by arrows inside the Pst I restriction map of wheat ctDNA. Wheat ctDNA fragments used as hybridization probes were a gift from C. M. Bowman and T. A. Dyer, Plant Breeding Institute, Cambridge. Barley ctDNA fragments used as hybridization probes are identified on the map by the prefix pHvc. The positions of the 235 and 16s ribosomal RNA genes within the large inverted repeat region (IR) are shown (Bowman et al., 1981). Only one of the two possible orientations of the intervening single copy regions is shown. The locations of the genes encoding cytochrome f (Willey et al., 1984) the large subunit of ribulose bisphosphate carboxylase (LS) (Bowman et al., 1981), and the alpha, beta, epsilon, and proton translocating III subunits of ATP synthase are shown (Howe et al., 1982; Howe et al., 1983). The 32,000 dalton chloroplast membrane polypeptide was located by comparison with its location on maize ctDNA (Paulsen, 1983; Bedbrook et al., 1978).

bisphosphate carboxylase (Bowman et al., 1981) the beta and epsilon subunits of ATP synthase (Howe et al., 1982) and most of the cytochrome f gene (Willey et al., 1984). This fragment was underrepresented in Barn HI digestions of total DNA from a number of different cloned albino plants (Figure 3A, lanes l-8). In most cases, pTac B2 hybridized to Barn HI fragments that were smaller than the 9.6 kb Barn HI fragment that was present in Barn HI digestions of total DNA from green leaf tissue (Figure 3A, lane G). These smaller fragments could be digestion products of nuclear DNA mitochondrial DNA or ctDNA.

Our total DNA extract from sterile root tissue of green plants showed more than a 50-fold reduction in ctDNA levels relative to total DNA from wheat albino clone 6 (see section 6; levels of ctDNA in albino clone 6). At the reduced levels of ctDNA present in total DNA from root tissue, the 9.6 kb Barn HI fragment represented the major fragment hybridizing to pTac B2 in Barn HI digestions of total DNA from roots (Figure 4, lane 1). Furthermore, no major altered fragments were visible in Eco RI and Xho I digestions of total root DNA (Figure 4, lanes 3 and 5). This result strongly suggests that the major bands that hybridize to pTac B2 in Barn HI digestions of total DNA from albino plants (Figure 3A, lanes MIX and lanes l-8) are not the result of homologous sequences present in nuclear or mitochondrial DNA. By elimination, we suggest that these major bands are derived from altered ctDNA molecules present in the proplastids of albino pollen plants. Firm evidence will only be obtained once ctDNA can be isolated (and cloned) from the proplastids of albino pollen plants. At present, methods have not been developed for isolating proplastids from limited amounts of tissue (5 g or less) in the quantities required for ctDNA extraction.

CI&oroplast Deletions and Maternal Inheritance

A G MIX 1 2 3 4 5 6 7 8 B GMIX~ 234567%

3.20, 3.20’ v

kb

10.7, 8.0 ’

6.9,

5.15,

Figure 3. Southern Blot Analysis of Wheat ctDNA from Green Tissue (G), A Population of Wheat Albino Plants (MIX), and Wheat Albino Clones 1-8

Total DNA was digested with Barn HI and the fragments separated on a 0.7% agarose gel. Fragments were transferred to nitrocellulose and hybridized with (A) 32P-labeled pTac 62 and (B) 32P-labeled pHvc B9. In (A), 5 pg of digested total DNA was loaded on lanes MIX to 8, and 0.5 pg loaded on lane G (1 cm wide slots). Half of these quantities were used in (B) (0.5 cm wide slots). Fragment sizes were estimated from marker restriction fragments of lambda DNA.

Kb

-13

-9.6

-3-7

-2.6

- 2.2

Figure 4. Southern Blot Analysis of ctDNA from Green Leaf and Sterile Root Tissue from Green Plants

Two hundred and fifty nanograms of total DNA from root tissue and 2.5 ng of total DNA from green leaf tissue were digested with the enzymes indicated above each lane and the digestion products separated on an 0.8% agarose gel before being transferred to nitrocellulose and hybridized with nick-translated 3ZP-labeled pTac 82. Sizes of major hybridizing bands were estimated from marker restriction fragments.

Major ctDNA Molecules Present in Most Albino Pollen Plants Lack the 9.6 kb Barn HI Fragment In Barn HI digests of total DNA from green leaves (Figure 3A, lane G), pTac B2 hybridized strongly to a 9.6 kb

fragment and weakly to two fragments (8.0 and 5.15 kb) that hybridized to pHvc 89 (Figure 38, lane G). pHvc B9 contains a 5.15 kb Barn HI fragment of barley ctDNA. pHvc B9 hybridized to two distant regions of the wheat chloroplast genome because it contains sequences present in one of the large inverted repeat regions of ctDNA (see Figure 2). The cloned 9.6 kb Barn HI fragment of wheat ctDNA and the cloned 5.15 kb Barn HI fragment of barley ctDNA hybridized weakly to each other on Southern blots in reciprocal hybridization experiments (results not shown). This result suggests that the weak hybridization between pTac B2 and the 8.0 and 5.15 kb fragments in Figure 3A is due to a distant cross-homology in the wheat chloroplast genome (this was brought to our attention by C. M. Bowman, Plant Breeding Institute, Cambridge). We have also found a cross-hybridizing DNA sequence that is located at similar positions on the barley chloroplast genome. The product of the cross-hybridization reaction between cloned barley ctDNA restriction fragments has a melting temperature that is approximately 5°C lower than the melting temperature of the 5.15 kb Barn HI fragment (B9) of barley ctDNA (unpublished results).

The 5.15 kb Barn HI fragment was unaltered in size and was present as a major fragment in Barn HI digestions of DNA extracts from seven of the eight cloned albino plants (Figure 38). Because of the fortuitous cross-hybridization shown between pTac B2 and pHvc B9, this fragment was also visible when pTac B2 was used as a hybridization probe (Figure 3A). The unaltered 9.6 kb and 8.0 kb Barn HI fragments do not represent major ctDNA fragments in digestions of DNA extracts from albino plants (Figures 3A and 3B, lane MIX and lanes l-8). The underrepresentation of the 9.6 and 8.0 kb ctDNA fragments in digestion mixtures of DNA extracts from albino plants suggests that a large region of the chloroplast genome was deleted in the major class(es) of ctDNA molecule present in these plants. Large deletions that remove the 9.6 kb Barn HI fragment and that end in the 8.0 kb Barn HI fragment (see

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Figure 2) may be responsible for the generation of the 10.7, 3.2, and 6.9 kb fragments visible in the lanes marked 4, 5, and 8, respectively, in Figure 3B.

Mapping Altered ctDNA Molecules Present in Albino Pollen Plants Our preliminary investigations showed that total DNA from a population of albino pollen plants produced all the ctDNA restriction fragments present in digestions of total DNA from green leaf tissue. However, the stoichiometries of the fragments produced by the two DNA preparations were markedly different. These early results (not shown) suggested that the total DNA preparation from a population of albino plants contained a number of different types of altered ctDNA molecule. We were unable to map the individual classes of ctDNA molecule that were present in this heterogeneous ctDNA population. In an attempt to reduce the number of ctDNA molecules present in DNA extracts we prepared DNA from the leaves of single cloned albino plants.

Sufficient total DNA was extracted from five of the eight original albino clones used in Figure 3 (clones 3-7) to enable us to perform physical mapping studies of their chloroplast genomes. We used the Pst I restriction map of wheat ctDNA (Bowman et al., 1981) to map the structures of the ctDNA molecules present in these DNA preparations Pst I digests ctDNA completely but leaves the nuclear DNA largely undigested (Bowman and Dyer, 1982). This means that by using purified ctDNA as a nick-translated hybridization probe, most of the Pst I ctDNA fragments can be visualized in digestions of total DNA (see Figure 5). Contaminating nuclear DNA in the probe hybridizes predominantly to high molecular weight DNA at the top of the gel. Although a heterologous probe was used (barley ctDNA), all the wheat ctDNA Pst I fragments can be seen in digestions of DNA from green tissue (Figure 5, lane G); the section of the autoradiograph containing the 1.9 kb (PII) and 1.4 kb (P12) fragments is not shown in Figure 5. The background hybridization was higher in lanes 3-7 of Figure 5 because of an approximately 2-fold reduction in the amount of ctDNA in total DNA from albino tissue relative to total DNA from green tissue (see below). This background hybridization obscures any Pst I fragments of ctDNA that may be present in the high molecular weight region (-25 kb) of the gel. Large Pst I fragments were detected using cloned ctDNA restriction fragments as hybridization probes (see Table 1).

Map Locations of Major ctDNA Pst I Fragments in Digested Total DNA from Albino Plants Our aim was to assign a map location to all the Pst I fragments visible in Figure 5. This was accomplished by using cloned wheat and barley Pst I fragments of ctDNA (see Figure 2) as hybridization probes against filter-bound Pst I fragments of total DNA from cloned albino plants. The results of these experiments are summarized in Table 1. The number of different hybridization probes used against Pst I digestions of total DNA from a given albino

(2

Albino Leaf P \

G'3 4 5 6 7

kb

-15.8

2:+8 -1012

-4.6

-4.3

-2.6

Figure 5. Major Pst I ctDNA Fragments Present in Green Leaf (G) and Different Albino Leaf (3-7) Total DNA Extracts

Two and one half micrograms of total wheat DNA from albino leaves and 0.25 pg of total wheat DNA from green leaves were digested with Pst I, the digestion products separated on a 0.7% agarose gel and transferred to nitrocellulose and then hybridized with the 32P-labeled barley ctDNA. Frag- ment sizes of unaltered ctDNA fragments were taken from Bowman et al., 1981 and agreed well with sizes estimated from marker restriction fragments. Altered fragment sizes were estimated from marker restriction fragments.

plant was limited by the amount of total DNA extracted from that plant. Table 1 shows that all the Pst I fragments visible in Figure 5 hybridized to one or more of the cloned ctDNA fragments used as hybridization probes. All the Pst I fragments (including substoichiometric bands) visible in Figure 5 were called major fragments.

The data of Table 1 were used to produce physical maps of the ctDNA molecules present in three different cloned albino plants (see Figure 6). Mapping was compli- cated by two factors: first, by the large inverted repeat sequence of ctDNA, and second, by the presence of more than one type of major ctDNA molecule in total DNA from one albino plant. Albino Clone 6 The stoichiometry of bands present in lane 6 of Figure 5 shows that albino clone 6 contained only one major ctDNA molecule. The determination of its structure was straight- forward. We used the known linear order of Pst I restriction fragments in wild-type ctDNA to arrange unaltered (in size) Pst I ctDNA fragments remaining in digestions of total DNA

Chloroplast Deietlons and Maternal Inheritance 363

Table 1. Summary of Hybrldizatlons between Cloned ctDNA Fragments and DNA from Green and Albino (A3-A7) Leaves

Major Filter-Bound Pst I Fragments (kb)

Hybndlzatlon Probes Green Leaf A3 A4 A5 A6 A7

pHvc P2 33.0, 19.6 33.0m, 19.6 19.6, 15.8 19.6, 13.0 19.6 lO.Om

pHvc P3 33.0, 19.6 33.0m, 19.6 19.6, 15.8 19.6, 13.0 19.6 lO.Om

pTac P3 14.5 14.5, 3.6m 11.8, 10.2 14.5, 13.0 4.3 4.6, 2.6

pTac P4 12.6 12.6 Ab 12.6, 5.9m Ab NT

pTac P5 11 .O, 5.6 11 .O, 5.6 11 .O, 10.2. 5.6 11 .O. 5.6 4.3 NT

pTac P6 8.4 a.4 8.4 8.4 8.4 Ab

pTac P7 8.1 8.lm Ab 5.9m Ab NT

pTac P8 11 0. 5.6 11 .O, 5.6 11.0, 10.2, 5.6 11 .O, 5.6 4.3 Ab

pTac P9 5.3 5.3 5.3 5.3 Ab NT

pTac PlO 5.2 5.2 5.2 5.2 5.2 5.2

pTac 02 33.0,8.1, 1.9 33.0m, 8.lm, 1.9m Ab Ab Ab Ab

pTac P12 1.4 1.4 1.4 1.4 1.4 NT

m-Minor fragment that showed a 5-lo-fold reduction in hybridization intensity relative to other filter-bound fragments present and is not visible in Figure 5. Ab-Absence of a detectable hybrldizatlon slgnal. NT-Hybridlzatlon reaction was not tested.

C Figure 6. Pst I Restriction Maps of Major Deleted ctDNA Molecules

The deleted ctDNA molecules were found in total DNA from (A) albino clone 6, (B) albino clone 4, and (C) albino clone 5 (only one of the two possible orientations of single copy sequences is shown). The fragment numbering scheme of intact ctDNA (Bowman et al., 1981, also see Figure 2) has been used to number fragments present in these deleted ctDNA molecules. The position of DNA sequences present in the large inverted repeat of intact ctDNA are marked by arrows. Deleted derivatives of Pst I fragments are indicated by ’ and numbered according to the origin of sequences present in these fragments. In (A) the 4.3 kb fragment (P8’ or P5’ + P3’) is derived from the 14.5 kb (P3) and the 5.6 kb (P8) or 11 .O kb (P5) fragments (see text). Both pTac P5 and pTac P8 hybridized with equal intensity to the altered 10.2 and 4.3 kb fragments which suggests that the predominant hybridization is due to sequences common to both probes that lie in the inverted repeat.

from albino clone 6. ctDNA from clone 6 only produces four major unaltered Pst I fragments of 19.6 (P2), 8.4 (P6), 5.2 (PIO), and 1.4 kb (P12). The 19.6, 8.4, and 5.2 kb

fragments are visible in lane 6 of Figure 5. These fragments are placed in the order Pi O-P1 2-P2-P6 in accordance with their arrangement in wild-type ctDNA (see Figure 2). Pst I fragments of 14.5 (P3) and 5.6 kb (P8) normally lie adjacent to PI0 and P6, respectively. When the 14.5 (P3) and 5.6 kb (P8) fragments were used as hybridization probes, they both hybridized strongly to the same altered 4.3 kb fragment. This suggests that the 4.3 kb fragment joins the ends of the structure Pi O-PI 2-P2-P6 to each other to form a circular ctDNA map of 39 kb (see Figure 6A). Since pTac P8 contains part of the large inverted repeat sequence that is present in pTac P5 (see Figure 2), it is also possible that the 4.3 kb fragment is derived from the 11 .O kb (P5) Pst I fragment.

The proposed structure of the 39 kb ctDNA map is consistent with the 21 (S2), 7.2 (S5), 1.2 (S9), and 8.4 kb (S7’ + S8’) fragments (for map locations see Bowman et al., 1981) present in Sal I digestions of total DNA from clone 6 (results not shown). The presence of a 39 kb ctDNA molecule in albino clone 6 was supported by the detection of a large open-circular molecule (of unknown size) and a linear molecule of 35-45 kb that hybridized to pTac PI0 in undigested total DNA extracts from albino clone 6 fractionated by agarose gel electrophoresis (results not shown). The conformation of the linear molecule was suggested by its comigration with linear restriction fragments in gels of two different agarose concentrations run at different voltage gradients. We do not know whether the linear molecule is a conformational isomer of the open- circular molecule. Albino Clones 4 and 5 The circular restriction map of the 39 kb ctDNA molecule present in albino clone 6 provides a basis for proposing circular DNA maps for major ctDNA molecules present in albino clones 4 and 5. In both cases, a rearranged fragment (10.2 kb in clone 4 and 13.0 kb in clone 5) was

Cell 364

detected that hybridized strongly to cloned ctDNA fragments from distant regions of the chloroplast genome (see Table 1). The uniformly higher intensities of the 19.6, 13.0, 11 .O, 8.4, 5.6, 5.3, and 5.2 kb Pst I fragments in digestions of DNA from albino clone 5 (Figure 5, lane 5) is consistent with these fragments, originating from one molecule that has the structure shown in Figure 6C. The substoichiometric 14.5 and 12.6 kb Pst I fragments presumably represent another major (though not predominant) type of ctDNA molecule present in this plant. The 5.9 kb Pst I fragment in albino clone 5 that hybridized to a mixed probe of pTac P4 and pTac P7 (Table 1) was present at lower levels than the 14.5 and 12.6 kb Pst I fragments and could not be detected using purified ctDNA as a hybridization probe (Figure 5, lane 5). These results may indicate the presence of a number of minor rearranged ctDNA molecules (that all contain the 14.5 and 12.6 kb fragments) in albino clone 5.

Two equally convincing models explain the results obtained with wheat albino clone 4. Both models explain the presence of multiple fragments (some of which are unaltered in size) that hybridize to the cloned ctDNA fragments present in pTac P3, P5, and P8. The first model assumes that more than one type of major ctDNA molecule was present in this plant. The postulated structure of one of these molecules is shown in Figure 6B, other fragments including altered ones of 15.8 and 11.8 kb (Figure 5, lane 4) were assumed to arise from a different major rearranged ctDNA molecule. The second model assumes that all the fragments present in lane 4 of Figure 5 were from one major altered ctDNA molecule that had been derived from wild-type ctDNA by duplication and rearrangement. Albino C/one 7 The major types of ctDNA molecule present in albino clone 7 contain only two unaltered Pst I fragments of 5.2 (PIO) and 1.4 kb (P12) (see Figure 2). The 1.4 kb Pst I fragment was detected using purified ctDNA as a hybridization probe. Our inability to detect major deleted derivatives of the 19.6 kb Pst I fragment (P2) of ctDNA in digestions of total DNA from albino clone 7 may reflect the small size of the deleted derivative of P2 or the presence of a heterogeneous population of deleted ctDNA molecules that result from multiple deletion endpoints in P2. The latter sugges- tion is supported by the presence of a minor 10.0 kb Pst I fragment that hybridized to pHvc P2 and pHvc P3. The 4.6 and 2.6 kb fragments present in Pst I digestions of ctDNA from albino clone 7 may be derived from two different types of rearranged ctDNA molecule in which different lengths of the 14.5 kb fragment have been deleted. However, the possibility that they arise from one type of rearranged ctDNA molecule cannot be excluded. Albino C/one 3 ctDNA from albino clone 3 and green leaf tissue produce all the Pst I fragments present in intact ctDNA (see Table 1). However, the stoichiometries of fragments present in the two DNA preparations were different. The 33.0 (PI), 8.1 (P7), and 1.9 kb (Pl 1) Pst I fragments were underrepresented relative to the other Pst I fragments produced

in digestions of DNA from clone 3. We compared the relative intensities of hybridization of pTac B2 to the 5.15 (B9) and 9.6 kb (82) Barn HI fragments present in digestions of DNA from green leaf tissue (Figure 3A, lane G) with the relative hybridization intensities of these fragments in DNA from albino clone 3 (Figure 3A, lane 3). Densitom- eter tracings of these lanes showed that the intensity of the 9.6 kb Barn HI fragment was approximately 10% of the expected value in the lane containing DNA from albino clone 3. These results suggest that albino clone 3 contained a heterogeneous ctDNA population in which most ctDNA molecules are altered.

Unaltered ctDNA Pst I Fragments Remaining in Digests of DNA from Albino Plants Unaltered Pst I fragments remaining in digests of DNA from wheat albino plants 3-7 were consistent with the locations of unaltered Sal I and Sst I fragments present in digestion patterns generated by probing total DNA digests with barley ctDNA (results not shown). The resolution of the method used means that we cannot exclude the possibility that small internal changes, e.g., rearrangements, insertions, deletions, nucleotide substitutions, had taken place within restriction fragments of apparent unaltered size.

Levels of ctDNA in Albino Clone 6 We used pTac PI2 as a hybridization probe against dilutions of a known amount of filter-bound Pst I digested pTac PI2 that were adjacent to lanes containing known amounts of Pst I digested total DNA from albino clone 6 and green leaf tissue. Using size estimates of 39 kb and 135 kb (Bowman et al., 1981) for the sizes of the chloroplast genomes in albino clone 6 and green leaf tissue, respectively, we were able to calculate the amount (by weight) of ctDNA in total DNA from clone 6 and our green leaf total DNA preparation. In addition, using a value of 18.9 pg as the haploid nuclear DNA content of T. aestivum (Davies and Rees, 1975) we were able to calculate the number of ctDNA molecules present per haploid nucleus, shown in Table 2.

Discussion

The Chloroplast Genome of Albino Pollen Plants Our studies showed that most of the ctDNA molecules in wheat albino plants regenerated from pollen grains were deficient in large regions of the chloroplast genome. In contrast, we have not been able to detect any alterations in ctDNA from green pollen plants of barley (results not

Table 2. Comparison of ctDNA Levels in Leaves from Normal Green Plants and Albino Clone 6

Tissue

Green Leaf

Albino Leaf

Size ctDNA ctDNA/Total DNA Copies ctDNA/

W % by Weight Haploid Genome

135 10 + 1.2 14,000 + 1,700

39 1.7 + 0.3 7,500 f 1,300

Chloroplast Deletions and Maternal Inheritance 365

shown). Restriction maps of three deleted derivatives of ctDNA present in different wheat albino pollen plants were proposed. In each case, deletions extend from a location within, or close to, the large inverted repeat to the 14.5 kb Pst I fragment (P3). More extensive deletions produce altered ctDNA molecules that have probably lost both large inverted repeat sequences, together with the intervening small single copy region and most of the large single copy region. These ctDNA molecules retain the region of the chloroplast genome that hybridizes to pTac Pi 0 and P12. This suggests that this region of the wheat chloroplast genome contains at least one functional origin of replication

Deletions in Organelle DNA: Proposed Mechanism for Deletion of ctDNA in Albino Pollen Plants Grossly deleted forms of organelle DNA have also been found in the chloroplasts of algae (Heizmann et al., 1981; Hussein et al., 1982; Myers et al., 1982) and the mitochondria of fungi (Locker et al., 1979; Cummings et al., 1979; Betrand et al., 1980). The type of deletions encoun- tered in each case reflect the sequence organization of the different organelle genomes and also the conditions under which the lesions were formed. Of the cited examples, Chlamydomonas ctDNA (which has the large inverted repeat organization, Rochaix, 1978) resembles wheat ctDNA the most. Deleted ctDNA molecules in 5fluoro- deoxyuridine (without X-rays) treated Chlamydomonas cells contain deletions that extend into the large inverted repeat mainly from one of the four bordering single copy sequences (Myers et al., 1982). This resembles the situa- tion in albino pollen plants where one of the two Barn HI fragments containing the large single copy region/large inverted repeat border was predominantly deleted.

Models involving recombination have been invoked to explain deletions in ctDNA and mitochondrial DNA (Ravel- Chapuis et al., 1984; Flamant et al., 1984; Bernardi, 1979). The mitochondrial DNA sequences retained in petite mutants of yeast appear to result from recombination between a short stretch of sequence homology in the wild-type mitochondrial genome (Gaillard et al., 1980). Small repeated sequences have been widely implicated in DNA rearrangements in both procaryotes and eucaryotes (Landy and Ross, 1977; Efstratiadis et al., 1980; Albertini et al., 1982; Zurawski et al., 1984; for further examples see Shapiro, 1983). The result of recombination between hypothetical short directly repeated DNA sequences in wheat ctDNA is shown in Figure 7A. If one of the products in Figure 7A (step 4) is lost through inability to replicate or vegetative segregation (Birky, 1978) the cell and its de- scendants will contain deleted ctDNA. Palmer and Thomp- son (1983) have suggested a recombination model to account for evolutionary changes in ctDNA. Large rearrangements in ctDNA (with the inverted repeat organization) evolution are restricted to inversions in the large single copy region; there are no major rearrangements that mix large and small single copy regions (Palmer and Thomp- son, 1982). The pathway in Figure 6B suggests why

6

0

A

B s d

7

co

a 0

Frgure 7. Production of Deleted ctDNA Molecules by Recombinatron

A srngle recombination event is shown between two hypothetical small repeats (+) flanked by small single copy sequences (a) and (b). and large copy sequences (c) and (d). In (A) the short repeat sequences are present in drrect orientation with respect to each other. In (B) the short repeat sequences are inversely oriented. The location of the large inverted repeat region contarnrng the ribosomal genes (not shown) are marked by heavy arrows. In (B) two recombrnation events, one rnvolvtng the hypothetical short inverted repeat (steps 1-4) and the other involving the large inverted repeat ragron of ctDNA (steps 5-7) are requrred to produce smaller deleted forms of ctDNA. Steps 5-7 could occur relatrvely quickly because when the large repeats of ctDNA are present in an Inverted onentatron, recombinatron between them is probably frequent on a developmental trme scale (Palmer, 1983).

inversions (the predominant evolutionary changes in ctDNA) cannot mix large and small single copy sequences. This pathway produces deleted molecules that resemble those found in albino pollen plants and which would be

Ct?ll 366

lethal under normal conditions. Furthermore, we can suggest that the inverted repeat structure does not confer stability on the chloroplast genome (Palmer and Thomp- son, 1982) rather most rearrangements will result in the formation of defective molecules. This model therefore suggests that the pathway responsible for evolutionary changes in ctDNA may also be responsible for the production of deleted ctDNA in albino pollen plants.

At What Stage Are the ctDNA Deletion Events Taking Place? We have detected deleted ctDNA molecules in total DNA from callus produced by barley anther culture (results not shown). Segregation of altered chloroplast genomes produced before or during the callus stage of anther culture would be expected in the following cellular divisions (Birky, 1978). During this phase there may be selective forces ensuring the preferential amplification and therefore sur- vival of particular chloroplast genomes. This implies that the altered ctDNA molecules present in albino pollen plants represent a subset of a larger spectrum of altered ctDNA molecules associated with anther culture.

Is ctDNA Degradation in the Male Gametophyte Responsible for Maternal Inheritance of Chloroplasts? ctDNA deletions in albino pollen plants may be the result of normal pollen development or a consequence of the culture methods used. Organelle alteration in the genera- tive cell has been proposed as a possible mechanism for maternal inheritance of chloroplasts (Vaughn et al., 1980). This process may be responsible for the ctDNA deletions that are observed in albino pollen plants of wheat and barley (results not shown). Albino plants would be derived from a cell lineage that had undergone organelle alteration. We are currently testing this hypothesis by looking for the presence of altered ctDNA molecules in mature pollen.

Experimental Procedures

Plant Material The variety of wheat used was Triticum aestivum cv Sappo. Methods used to produce plants by anther culture are described elsewhere (Huang and Sunderland, 1982). Clones were propagated vegetatively from regenerants derived from different pollen calluses. The following pairs of cloned albino plants were regenerated from separate calluses originating from the same culture vessel (see Figure 1, step C); (1) clones 2 and 3, (2) clones 5 and 6, and (3) clones 7 and 8. Albino plants were a gift from L. Demetriou and B. Huang. These were grown aseptically in jars containing half-strength Murashige and Skoog medium (1962) supplemented with 2% W/V sucrose.

Green tissue was obtained from 7-14 day old seedlings grown in compost in a heated greenhouse. Root tissue was cut from the roots of four plants grown on a half-strength Murashige and Skoog medium from seeds that were surface-sterilized with a solution containing 2% v/v sodium hypochlorite.

ctDNA Extractions ctDNA was isolated by the nonaqueous method of Bowman and Dyer (1982). DNA from lysed chloroplasts was purified on CsCl/ethidium bromide density gradients.

Total DNA Extractions The method described by Cullis (1979) was used for DNA extractrons from green tissue and populations of albino plants. However a-amylase was not used. A modified procedure was used to extract DNA from limited amounts of tissue. Leaves or roots were first ground in liquid nitrogen, the RNAase treatment was omitted, and the DNA purified by preparative CsCl centrifu- gation Immediately after pronase digestion. DNA concentrations were determined from OD readings taken at 260 nm.

Restriction Enzymes Pst I and Sst I were purchased from BRL(UK) Ltd, Cambridge, England. Barn HI, Sal I, and Sal PI (an isoschizomer of Pst I, Chater et al., 1977) were a gift from P. Dlckerson (of the John lnnes Institute). Digestions were performed in standard assay buffers.

Electrophoresis, Transfer, and Hybridization DNA restriction fragments were separated on vertical 0.7% or 0.8% agarose gels using 40 mM Tris-acetate (pH 7.9), 5 mM sodium acetate, 1 mM EDTA as the gel buffer. Molecular weight marker fragments were obtained by digesting lambda DNA with Hind Ill, Hind Ill plus Eco RI and Xho I. The lambda DNA sequence was used to calculate the sizes of restriction fragments (Sanger et al., 1982). DNA fragment sizes were estimated according to the method of Southern (1979) using the nonlinear regressron analysis of Duggleby (1981). Gels were stained in ethidium bromide (1 pg/ ml) and large fragments nicked with a short-wave UV source (254 nm) prior to transfer. DNA was transferred to nitrocellulose by the method of Southern (1975). Hybridization conditions have been described (Goldsbrough et al., 1981). The prehybridization solution contained denatured total DNA from roots (l-5 pg/ml) when ctDNA preparations were used as probes. In some cases the rate of hybridization was increased by adding de&an sulphate (Wahl et al., 1979; Figures 3A, 38, 4, and 5).

Filters were washed in 2X SSC, 0.1% SDS at 65°C for a minimum of 4 hr (IX SSC = 0.15 M sodium chloride, 15 mM trisodium citrate).

Hybridization was detected by exposing filters to Fuji Rx X-Ray film. In most cases sensitivity of the film was increased by prefogging the film and exposing it at -70°C (Laskey and Mills, 1975) in llford cassettes containing intensrfying screens,

Rehybridization Previously hybridized probes were washed off according to the method of Thomas (1980).

Recombinant DNA Clones pTac plasmids were a kind gift from C. M. Bowman and T. A. Dyer (Plant Breeding Institute, Cambridge). pHvc plasmids constructed here contained barley ctDNA restriction fragments ligated into the Barn HI or Pst I site of pAT 153.

Supercooled plasmids were purified from cleared lysates (Clewell and Helrnski, 1970) on preparative caesium chloride/ethidium bromide density gradients (Radloff et al., 1967).

Acknowledgments

We thank Dr. C. M. Bowman and Dr. T. A. Dyer for their generous grft of pTac plasmids and useful comments, P. Scott and K. Glencross for photographic work, and B. A. Bowen, D. R. Davies, J. M. Dunwell, E. A. Mudd, and K. A. Pyke for their advice on the manuscript. A. D. was supported by an Agricultural Research Council Studentship.

The costs of publicatron of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Recerved July 11, 1984; revised September 11, 1984

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Chloroplast DNA deletions associated with wheat plants regenerated from pollen: possible basis for maternal inheritance of chloroplasts