Plant MolecularBiology7: 143-149, 1986 © MartinusNijhoffPublishers, Dordrecht - Printedin the Netherlands

143

Short communication Divergence of chloroplast gene organization in three legumes: Pisum sativum, Vicia faba and Phaseolus vulgaris Edwin J. Crouse, 1,2,* Mfika Mubumbila, 2 Bjarn M. Stummann, 1 Gerhard Bookjans, 1,3 Christine Michalowski,4 Hans J. Bohnert,4 Jacques-Henry Weil2 & Knud W. Henningsen 1

1The Royal Veterinary and Agricultural University, Department of Genetics, Biilowsvej 13, DK-1870 Copenhagen V, Denmark 2Institut de Biologie Mol6culaire et Cellulaire du CNRS, Universitd Louis Pasteur, 15 rue Descartes, F-67084 Strasbourg Cedex, France 3Present address: Department of Agronomy, College of Agriculture, University of Kentucky, Lexington, K Y 40546-0091, U.S.A. 4University Department of Biochemistry, BioSciences West, University of Arizona, Tucson, A Z 85721, U.S.A. Keywords: chloroplast DNA, molecular evolution, polypeptide genes, tRNAs, tRNA genes

Summary Isolated chloroplasts from Pisum sativum were found to contain at least 32 tRNA species. Hybridization of in vitro labeled, identified, chloroplast tRNAs to Pisum chloroplast DNA fragments revealed the locations of the tRNA genes on the circular chloroplast genome. Comparison of this gene map to the maps of Vicia faba and Phaseolus vulgaris showed that the chloroplast genomes of Pisum and Phaseolus are otherwise more closely related than either genome is to the chloroplast genome of Vicia. Furthermore, the results suggest how possible recombination events could be involved in the evolution of these three closely related, but divergent, chloroplast genomes.

Introduction Analysis of higher plant chloroplast DNAs (cpDNAs) by electron microscopy and/or restriction endonuclease cleavage revealed that most plastid genomes contain a large region, which is present twice within the molecule, but in an invertcd orientation [1, 7, 8, 27]. Each repeated region contains a set of rRNA genes. Exceptions to this general organization have been reported for some members of the Leguminosae family. For instance, in contrast to the larger cpDNA of Phaseolus vulgaris (common bean), which h~as the invertedrepeat structures, the smaller cpDNAs of Pisum

sativum (pea) and Vicia faba (broad bean) do not have the inverted repeat regions and contain only one set of rRNA genes. In previous studies on Vicia [16] and Phaseolus [17], at least 31 tRNA genes were mapped on these two chloroplast genomes. In this report, the number and location of the tRNA genes on the Pisum chloroplast genome are compared to the corresponding data from Vicia and Phaseolus. The positions of the genes coding for the a, ~, and ~ subunits of the coupling factor, the large subunit of ribulose-l,5-bisphosphate carboxylase and the herbicide binding 32 kDa polypeptide of photosystem II are also compared.

*Allcorrespondenceto: Dr E. J. Crouse, IBMCdu CNRS, 15 rue Descartes, F-67084StrasbourgCedex,Franc~ Tel. (33)-88.61.02.02.

144 Materials and m e t h o d s

Results and discussion

Chloroplasts were isolated from the leaf material by differential centrifugation [2]. After extraction of the nucleic acids, cpDNA was recovered by Sephacryl S-1000 column chromatography [2] and cleaved with restriction endonucleases. The DNA fragments produced were separated in horizontal agarose slab gels by electrophoresis and blotted onto nitrocellulose filters. Chloroplast tRNAs were recovered from the nucleic acid extract by DEAE column chromatography, fractionated by two-dimensional polyacrylamide gel electrophoresis, extracted from the gel, and identified by aminoacylation [3]. Individual tRNA species were treated with snake venom phosphodiesterase to remove the terminal nucleotide(s) of the -CCA end, enzymatically labeled at the 3' end [25] in the presence of (ot-32p)ATP, CTP and yeast tRNA nucleotidyl transferase [23], and then hybridized to filter immobilized cpDNA fragments using the conditions described in [16, 17]. Five polypeptide gene probes, prepared from cloned cpDNA fragments of Spinacia oleracea, were nick translated to high specific activities and also used for hybridization with cpDNA fragments.

Fractionation and identification of Pisum chloroplast tRNAs Pisum tRNAs were in vitro labeled and then fractionated by two-dimensional polyacrylamide gel electrophoresis. Methylene blue staining of the gel visualized the various RNA species, whereas autoradiography revealed the presence of 32 tRNAcontaining spots. After electrophoretic fractionation of unlabeled chloroplast tRNAs and extraction from the gel, the tRNAs were identified by aminoacylation. A total of 19 Pisum tRNAs for 11 amino acids were identified (Fig. 1 and Table 1). At least 13 Pisum tRNAs have not been identified, although they were observed in the gels because they can be labeled by the yeast tRNA nucleotidyl trans-

I dim I

0 O r, 5er2

Oel2(~gO~O000 Os:!iOLeulTr o

o0S.

0

Val2~~''O Ash,2 ~'~O,,.%Asnl

0

OU0

0

u

o oo OArg

aoq°1 O

Ile2

Fig. 1. Fractionationof Pisum chloroplast tRNAs by two-dimensionalpolyacrylamidegel electrophoresis. Diagrammaticrepresentationof methyleneblue stained gels after electrophoretic separation of chloroplast tRNAs using 1()¢/0poiyacrylamide/4 M urea for 40 h at 450 V in the first dimensionand 20°/o polyacrylamide/4M urea for 140 h at 350 V in the second dimension.The aminoacid acceptedby each identifiedtRNA is indicated.Our failureto identifysometRNAs may be due to the inactivation of these tRNAs during electrophoresisand/or to the inactivationof the correspondingaminoacyl-tRNAsynthetases.

145

Table I. Identified chloroplast tRNAs and mapped chloroplast tRNA genes of Pisum sativum. tRNA specific for:

No. of isoacceptors identified

No. of genes mapped

Ala Arg Asn Asp Cys Glu Gin Gly His Ile Leu Lys Met Phe Pro Ser Thr Trp Tyr Val

1 1 2

1a 2a Ia Ib

-

1b

1 2 3 l

1 2

1 or 1 1~ 3 to I 2 or 1 1 3 to 1d 1 1b 2~

Total

19

25 to 41

2 3

2c 10~ 3~ 10~

a One gene has been sequenced [G. Bookjans, unpublished data]. b The gene has been sequenced [22]. c One gene has been sequenced [J. Lehmbeck, unpublished data]. d The gene has been sequenced [O. Rasmussen, unpublished datal.

zation experiments were performed with 27 chloroplast tRNAs from Phaseolus, with chloroplast t R N A Ala from Zea mays and with chloroplast tRNA°Yl, tRNA°Y2 and tRNAWal from Spinacia oleracea. The heterologous hybridizations can be used to m a p t R N A genes on the c p D N A of Pisum because a high degree of homology exists a m o n g the same chloroplast t R N A species of various higher plants [15]. A minimum number of 25 genes, coding for tRNAs corresponding to 18 amino acids (Table 1), and three unidentified genes, were m a p p e d on the Pisum chloroplast genome (Fig. 2). Genes for t R N A cys and t R N A ° n have not been mapped; however, chloroplast genomes are known to encode genes for tRNAs corresponding to all 20 amino acids [7, 8]. Furthermore, the genes for t R N A cys o f Spinacia oleracea [9] and t R N A GIn o f Vicia faba [26] have been sequenced. D N A probes for five polypeptide genes from the chloroplast genome o f Spinacia oleracea were prepared from cloned D N A fragments (Table 2) and hybridized to c p D N A fragments. All probes comprised part of the coding region; some contained part of the 3' region, but none contained the 5' (upstream promoter) region to avoid crosshybridization between different genes with similar control regions. The inferred locations of the genes are shown in Fig. 3.

Comparison of the chloroplast gene maps of Pisum, Vicia and Phaseolus

ferase reaction which is specific for tRNAs. The total number of Pisum chloroplast tRNAs (at least 32) is approximately the same number found for Vicia [16] and Phaseolus [17], and approximately equal to the minimum number o f tRNAs, required to read all 61 sense codons, according to the wobble hypothesis [6]. Recently, 36 R N A species were detected upon two-dimensional electrophoretic separation of Pisum chloroplast 4S RNAs and genes for about 30 of these unidentified R N A species could be m a p p e d on the chloroplast genome [5].

Mapping of chloroplast genes For the mapping of Pisum chloroplast t R N A genes, in vitro labeled, identified, chloroplast tRNAs were hybridized to nitrocellulose blots of the fractionated D N A fragments. In addition to the homologous hybridizations, heterologous hybridi-

Comparison of the chloroplast gene maps of Pisum, Vicia and Phaseolus (Fig. 3) reveals the existence of the same group of t R N A genes in the vicinity of the r R N A genes: trnV2-16SrDNA-trnI2trnA-23SrDNA-trnN. Other c o m m o n groups of t R N A genes are trrff-trnY, trnH-trnK, and trnLtrnF-trnS-trnM2. Another cluster includes genes for four polypeptides. Unlike most other higher plant chloroplast genomes [1, 27], the rbcL, atpB and atpE genes m a p near the psbA gene (Fig. 3), This cluster is located about 40 to 60 kilobase pairs from the atpA gene. Similar results on the mapping of these five polypeptide genes were reported for Pisum [10, 20] and Vicia [11, 20, 24], but no data is available for Phaseolus. Despite the presence of similar groups of genes, several differences also exist showing that these three genomes are distinct. For example, in Phaseo-

146

t rr/ ,o

~.to Y"

trnM1

Fig. 2. Location of tRNA genes on the circular map of Pisum cpDNA. The positions of the restriction endonuclease cleavage sites are based on data from [4, 19]: o, SalI; v, PstI; e, SmaI; ¢, XhoI. Gene nomenclature for an unidentified tRNA gene is indicated by trnX and for identified tRNA gene by trn followed by the corresponding amino acid one letter code and the number of the isoacceptor.

Table 2. Polypeptide gene probes from the chloroplast genome of Spinacia oleracea used for heterologous hybridizations with cpDNA fragments. Gene

Gene product

Probe

Reference

atpA

c¢ subunit of CF~

1200 base pairs HindIII-SmaI

W. Bottomley, per5. commfin.

atpB atpE rbcL

~ subunit of CF= ~ subunit of CFi large subunit of ribulose:l,5bisphosphate carboxylase 32 kDa herbicide-binding PSII polypeptide

1627 base pairs ClaI-EcoRI 431 base pairs EcoRI-XbaI 1750 base pairs EcoRI

[29] [29] [30]

520 base pairs HindIII-XbaI

[28]

psbA

147

I

T1

L2

16.2

]lI2A

N21

19

L3IN2Az2t I 9.7

12.8

11

L2

172

K

s3

8.5

113

G2

T

~,

,

71 11 7

/(V1)

~..

5.6

I0

st 13

b.. 11

Ol or

Fig. 3. Rearrangements within Pisum cpDNA and within Vicia cpDNA relative to Phaseolus cpDNA.

Linear maps of all three circular chloroplast genomes are shown. The positions of the restriction sites are based on data from [4, 19] for the Pisum cpDNA, on data from [11, 12, 16, 20] for the Vicia cpDNA, and on data from [17, 21] for the Phaseolus cpDNA. Restriction site symbols: o, Sail; v , KpnI; v , PstI. The tRNA genes are indicated by the one letter symbols for the corresponding amino acids ('X' indicates an unidentified gene). The l~olypeptide genes have been indicated by sinile greek letters: c~ = atpA; B = atpB; e = atpE; h = rbcL; ~ = p s b A . Only one of the two populations of Phaseolus cpDNA [17, 18] is shown. The two populations differ in the orientation of the single-copy regions. Numbers below the Phaseolus map indicate the size of the PstI fragments in kilobase pairs.

lus, trnL1, trnL2 and trnL3 are located, respectively, in the large single-copy region, the repeated regions and the small single-copy region, whereas these genes are grouped together in the chloroplast genomes of Pisum and Vicia. Vicia has at least one group with all three genes and another group with only trnL1 and trnL2, whereas Pisum appears to have up to three groups comprising the genes (or possibly pseudo-genes) for all three isoacceptors. In addition, Vicia and Phaseolus have one copy oi the genes trnSl, trnS2 and trnS3, whereas Pisum has up to three groups of these genes (or pseudo-genes). Furthermore, the Vicia genome appears to have a higher copy number for the genes of tRNAGIyl, tRNAGIY2, tRNA pr°, tRNA Thr and tRNA Tyr. The copy number of other tRNA genes also varies. For instance, except for the trnL2 genes, the two copies of the tRNA genes found in the inverted-repeat regions of Phaseolus are reduced to

one copy in both Pisum and Vicia, indicating that a double set of these genes are not necessary for chloroplast function in the latter two plants. The total number of chloroplast tRNA genes, corresponding to all 20 amino acids, could be about the same (ca. 42 genes) in all three legumes. From a previous study on the number of Pisum chloroplast tRNA genes, approximately 42 genes were estimated by saturation-hybridization of total chloroplast tRNA to total chloroplast DNA [13, 14]. The reason for having a higher copy number of some tRNA genes is not known. Although one might be tempted to consider that the chloroplast genomes of Pisum and Vicia are closely related because both have only one rDNA unit, the genome of Pisum appears closer to that of Phaseolus when one considers the overall organization of tRNA genes. For example, near the Pisum rDNA unit (Fig. 3), one finds trnT,Y,G1,G2,S1,S3,

148 L1,F,M2,K,H (group A) just after trnV2,I2,A,R2,N (group B), whereas group A genes and group B genes of Phaseolus are separated. This organization of tRNA genes is not apparent in the case of the Vicia chloroplast genome. It should he pointed out that the Pisum tRNA genes of group A are flanked by the genes or pseudogenes for tRNA I~u. If these repeated tRNA t~u genes or pseudogenes have an opposite polarity, one can anticipate that the presence of these redundam genes (or a nearby specific sequence among these gene groups), located in different parts of the circular cpDNA molecule, could allow for the inversion of the intervening region through intramolecular recombination. With the presence of reiterated tRNA genes or pseudogenes, one can envisage how a chloroplast genome such as that of Pisum or Vicia could possibly be 'scrambled', relative to the Phaseolus genome, during evolution.

Acknowledgements The authors are grateful to Dr Gi6g6 (IBMC) for the yeast tRNA nucleotidyl transferase and tO Dr Palmer (University of Michigan) for his Pst-clones of Spinacia oleracea cpDNA. This study was SUl~ ported by grants from the Danish Agricultural and Veterinary Research Council to K.W.H. and the Deutsche Forschungsgemeinschaft to H.J.B:, and an EMBO long term fellowship to G.B.

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Received 7 August 1985; in revised form 12 May 1986; accepted 21 May 1986.