J. Plant Biochemistry & Biotechnology Vol.

1, 1-4, January 1992

Chloroplast Genome of Vigna aconitifolia and Localization

of Certain Photosynthesis-related Genes

N Y Kelkar1*, A K Tyagi2 and S C Maheshwari1, 2

1
Department of Botany, University of Delhi, Delhi-110 007
2
Department of Plant Molecular Biology, University of Delhi, South Campus, New Delhi-110 021, India

The restriction analysis of chloroplast genome of Vigna aconitifolia has revealed that it is about 150 kb in size, similar
to V. radiata. The restriction pattern of chloroplast DNA (cpDNA) for Pst I is also the same from both the species, but
restriction fragment length polymorphism is observed in cases of Kpn I and Sstl. These differences in the restriction patterns
have arisen because of the occurrence of different restriction sites in the chloroplast genome of V. aconitifolia. A restriction
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map of cpDNA for V. aconitifolia has been prepared on the basis of these observations. Furthermore, seven genes (psbA,
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psbB, psbC, psbD, psaA, psaB and rbcL) — coding for polypeptides of photosystems I and Il as well as the large subunit
of ribulose 1,5-bisphosphate carboxylase/oxygenase — have been localized on the Pst I - and Kpn I - generated restriction
fragments of V. aconitifolia with the help of heterologous gene-specific probes and their relative position on the restriction
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map is presented. The gene organization supports the view that an inversion of about 50 kb has occurred in Vigna cpDNA
as compared to other species.

Key words : chloroplast genome, photosynthesis-related genes, Vigna aconitifolia.

Chloroplast, the seat of photosynthesis in plants,
contains its own DNA ranging in size f r o m about
120-220 kb. The restriction maps of cpDNAs from a
number of plants have been prepared to characterize
cpDNAs and to understand the evolutionary
relationships (1). Furthermore, the cpDNA has been
shown to harbour genes for 4 rRNAs, 30 tRNAs and
about 60 proteins, in addition to about 20 open reading
frames whose products are not yet identified (2, 3).
Such studies have not only paved the way to identify
new components for chloroplast function but also
formed the basis for studies on gene organization and
expression.
The present work involves characterization of cpDNA
of Vigna aconitifolia, a drought resistant seed legume,
with the help of restriction enzymes Kpn I, Pst I, and Sst
I. in addition seven chloroplast-encoded genes (psbA,
psbB, psbC, psbD, psaA, psaB, and rbcL) related to pho-
tosynthesis have been localized on individual restriction
fragments and arranged on a restriction map for Kpn I
and Pst I.
Materials and Methods
Isolation of chloroplast DNA—Chloroplasts were
isolated from fresh leaves of 7-day-old seedlings of
Vigna species, raised on 3 cm thick absorbent cotton
pad at 28 ± 2°C and 12 W/m2 light according to Palmer
'Corresponding author
(4) with certain modifications (5). This involves use of
0.4 M sucrose in grinding buffer and purification of
chloroplasts on a three-step-gradient. The cpDNA
from lysed chloroplasts was purified on a cesium
chloride/ethidium bromide gradient as described by
Palmer (4) but with increased concentration of CsCl
(2.1 g/ml lysate).
Isolation of plasmids, restriction analysis and
Southern hybridization—Recombinant plasmids
containing gene-specific probes were isolated from
Escherichia coli cells employing the procedure of
Birnboim and Doly (6). For restriction analysis of cpDNA
and plasmids, manufacturer's specifications for each
restriction endonuclease were followed. The DNA
samples were resolved on 0.9% agarose gels by
electrophoresis in the running buffer (0.04 M Tris-
sodium acetate, 1 mM EDTA at pH 8.0). Southern
hybridization was carried out employing either nitrocel-
lulose filters according to standard protocol (7) or
Hybond N + as per manufacturer's recommendations
(Amersham international, U.K.). Gene-specific probes
(Table 1), prepared from t h e recombinant plasmids
and radiolabelled with [α - 32P] dCTP employing a
multiprime labelling system (Amarsham International,
U.K.), were used at levels of approximately 3 x 10 7
cpm (@ 3 x 10 8 cpm/µ.g DNA) per hybridization.
After suitable washings, f i l t e r s were covered with
Sar an wr ap and expos ed to X-r ay f i l m s for 24-
72 hr.
 2 J Plant Biochem Biotech

Table 1. Heterologous probes used for localization of genes

on the cpDNA fragments of V. aconitifolia

Gene Protein Probe size in bp Reference

(coding potential
in amino acids)

psbA 32 kD, D 1 789 (87-341) 13

psbB 51 kD 1160 (87-471) 14
psbC 44 kD 837 (52-330) 15
psbD 32 kD, D2 989 (upstream + 1-306) 15

psaA 68 kD 1100 (17-383) 16

psaB 66 kD 2100 (94-734) 17
rbcL LSU-RUBISCO 1800 (complete coding 18
region)
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Results and Discussion

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Restriction analysis of chloroplast DNA—In order to

characterize cpDNA of Vigna aconitifolia by restriction
analysis, it was digested with Kpn I, Pstl, and SstI whose
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recognition sites include purines as well as pyrimidines

but show a bias for C and G which are less prevalent
in cpDNA. These enzymes, therefore, produce fewer
fragments of a size range which could be easily resolved
by agarose gel electrophoresis. As the restriction map
of the cpDNA of V. radiata has already been established
(8), a comparison was made between the restriction
patterns of cpDNA of V. aconitifolia and V. radiata (Fig.
1). With Pst I, Kpn I, and Sst I, cpDNA of V. aconitifolia
produces 13, 10, and 14 bands whereas 13, 8, and 13
bands are obtained in case of V. radiata, respectively.
The bands with higher fluorescence contain double or
triple fragments of the same sizes, which makes 13, 11,
and 19 fragments for V. aconitifolia and 13, 10, and 18
fragments for V. radiata (Table 2). The size calculations
from the restriction fragments indicate that both the species
have cpDNA of about 150 kb. The restriction patterns
generated are exactly the same for both the species in
case of Pst I, but differ for Kpn I and Sst I. Thus, V.
aconitifolia lacks a fragment of 16.6 kb in comparison
to V. radiata for Kpn I and has two additional fragments
of 12.9 and 4.2 kb. Similarly, in Sst I digests, V, aconitifolia
does not contain a 16.6 kb fragment. Instead, two additional
fragments of 10.8 and 5.8 kb are observed.
It is well known that cpDNA of higher plants is highly
conserved in respect of components it codes for and the Fig. 1. Comparison of restriction patterns of V. aconitifolia (VA)
organization of the genes. The exact size of cpDNA, and V. radiata (VR). The cpDNA from both the species was
however, varies from 120-217 kb (1). Similarly, the num- digested with Pst I, Kpn I and Sst I. The sizes (kb) of DNA
ber of restriction sites as well as the sizes of restriction fragments based on λ DNA digest with Hind III are given on
fragments produced by a particular restriction endonu- the left. The same gel was photographed after 12 hr and 30
clease do differ among different taxa, even at intraspe- hr of electrophoresis and the best states of band separation are
cific levels (3). The variations in the restriction digests presented here. The upper part of the figure represents the state
of cpDNA would arise due to deletion or insertion of of large size DNA bands after 30 hr of electrophoresis; the lower
certain unique sequences or reorganization of DNA or part represents the state of small size DNA bands after 12 hr
due to point mutations. It is, therefore, possible that of electrophoresis.
 Chloroplast Genome and Photosynthesis-related Genes 3

Table 2. Comparison of the restriction fragments of V.aconitifolia generation of smaller fragments represents occurrence
(VA) and V. radiata (VR) on the basis of their sizes (kb). of additional restriction sites. Such restriction fragment
length polymorphism could be of great utility as markers
S. Pst I Kpn I Sst II
of chloroplasts in somatic hybrids, in particular, for analysis
No VA VR VA VR VA VR of cybrids (9,10).
1. 34.0 34.0 31.0 31.0 29.0 29.0 Localization of photosynthesis - related genes — As
2. 19.5 19.5 24.0 24.0 20.9 20.9 a first step towards characterization of the chloroplast
3. 17.4 17.4 24.0 24.0 17.3 17.8
genes, DNA fragments containing a particular gene were
identified by Southern hybridization involving heterolo-
4. 15.9 15.9 — 16.6 17.8 17.8
gous gene-specific probes (Table 1) and cpDNA frag-
5. 13.2 13.2 16.6 16.6 — 16.6
ments generated after digestion with two restriction en-
6. 11 2 11 2 155 15.5 10.8 —
donucleases (Kpn I and Pst I). In all, 7 genes have been
7. 9.6 9.6 12.9 — 10.0 10.0 localized out of which 4 (psbA, psbB, psbC, psbD) code
3. 7.9 7.9 8.4 8.4 8.1 8.1 for the polypeptides of PSII, 2 (psaA and psaB) for
9. 7.5 7.5 7.7 7.7 5.8 — polypeptides of PSI and one (rbcL) for the large subunit
10 7.0 7.0 4.4 4.4 5.2 5.2 of ribulose 1,5-bisphosphate carboxylase/oxygenase.
11. 5.8 5.8 4.2 — 4.2 4.2 Results of Southern hybridizations for localization of
12. 1.2 1.2 0.8 0.8 3.6 3.6 genes are shown in Fig. 2. The gene-specific probes for
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13. 1.1 1.1 3.6 3.6 individual genes hybridized to specific fragments. Since
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14. 2.8 2.8 the relative positions of these fragments can be deduced
15. 2.8 2.8
from a restriction map of cpDNA of V. aconitifolia based
on the information generated here and already available
16. 2.8 2.8
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on V. radiata (8), it is also possible to position various

17. 1.9 1.9
genes on the map (Fig. 3). The results indicate that the
18. 1.4 1.4
location of genes in V. aconitifolia is largely the same
19. 1.3 1.3 as that in V. radiata (11). Al! the genes, studied here,
20. 1.3 1.3 are located in the region called as LSC (large single
copy). On the left side of map for V. aconitifolia is pre-
Total 151 3 151.3 149.5 149.0 151.1 151.1

Fig. 2. Localization of chloroplast-encoded genes on cpDNA of V. aconitifolia, [A] cpDNA after digestion with restriction endonu-
cleases and separation on 1% agarose gel. [B] Autoradiograph showing bands after Southern hybridization of the gene-specific
probe with the cpDNA fragments. The restriction enzymes (K, Kpn I; P, Pst I) used and the genes localized are given at the top
of the lanes.
 4 J Plant Biochem Biotech
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Fig. 3. The position of various genes on the Pst I and Kpn I
generated physical map of V. aconitifolia which is based in part
also on the information concerning V. radiata cpDNA (7). The
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sizes (kb) of various fragments generated by Pst I and Kpn I
are also given. Abbreviations: IRA and IRB, inverted repeats A
and B; ISC, large single copy region; SSC, small single copy
region.
sent psbB within a 12.9 kb fragment instead of 16.6 kb
fragment of Kpn I, as in V. radiata. The smaller fragment
of V. aconitifolia has been generated due to creation of
a restriction site, thus allowing more precise location of
psbB. On the other side psbD and psbC, psaB and psaA
are located in a 8.1 kb region. An interesting feature of
V. radiata chloroplast genome is the close location of
rbcL and psbA unlike in most of the higher plants, e.g.
spinach and tobacco, where they are located about 50
kb away from each other. Such a change of gene organi-
zation of Vigna as well as Oenothera has been assigned
to an inversion of 50 kb fragment in the LSC region (12)
and is supported by data on gene localization in V.
aconitifolia.
Acknowledgements
We are grateful to Prof. R.G. Herrmann for providing us the
clones containing gene-specific DNA fragments. Thanks are also
due to the Department of Science and Technology for the funds
and to the University Grants Commission for the fellowship to
NYK.
Received 4 October, 1991
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