Direct measurement of the transfer rate of chloroplast DNA into the nucleus

Abstract

Gene transfer from the chloroplast to the nucleus has occurred over evolutionary time. Functional gene establishment in the nucleus is rare, but DNA transfer without functionality is presumably more frequent. Here, we measured directly the transfer rate of chloroplast DNA (cpDNA) into the nucleus of tobacco plants (Nicotiana tabacum). To visualize this process, a nucleus-specific neomycin phosphotransferase gene (neoSTLS2) was integrated into the chloroplast genome, and the transfer of cpDNA to the nucleus was detected by screening for kanamycin-resistant seedlings in progeny. A screen for kanamycin-resistant seedlings was conducted with about 250,000 progeny produced by fertilization of wild-type females with pollen from plants containing cp-neoSTLS2. Sixteen plants of independent origin were identified and their progenies showed stable inheritance of neoSTLS2, characteristic of nuclear genes. Thus, we provide a quantitative estimate of one transposition event in about 16,000 pollen grains for the frequency of transfer of cpDNA to the nucleus. In addition to its evident role in organellar evolution, transposition of cpDNA to the nucleus in tobacco occurs at a rate that must have significant consequences for existing nuclear genes.

Full text

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Supplementary Information accompanies the paper on Nature’s website
(çhttp://www.nature.com/nature).
Acknowledgements This work was supported by grants from the Biotechnology and Biological
Sciences Research Council (to E.J.L., I.N.R. and S.G.O.) and the Wellcome Trust (to S.G.O.).
We thank S. James and L. Lockhart for their help in some early analyses, and B. Dujon for
discussions.
Competing interests statement The authors declare that they have no competing financial
interests.
Correspondence and requests for materials should be addressed to S.G.O.
(e-mail: steve.oliver@man.ac.uk).
..............................................................
Direct measurement of the
transfer rate of chloroplast
DNA into the nucleus
Chun Y. Huang*, Michael A. Ayliffe†& Jeremy N. Timmis*
*Department of Molecular Biosciences, The University of Adelaide,
South Australia, 5005, Australia
†CSIRO Plant Industry, GPO Box 1600, Australian Capital Territory 2601,
Australia
.............................................................................................................................................................................
Gene transfer from the chloroplast to the nucleus has occurred
over evolutionary time
1
. Functional gene establishment in the
nucleus is rare, but DNA transfer without functionality is pre-
sumably more frequent. Here, we measured directly the transfer
rate of chloroplast DNA (cpDNA) into the nucleus of tobacco
plants (Nicotiana tabacum). To visualize this process, a nucleus-
specific neomycin phosphotransferase gene (neoSTLS2) was inte-
grated into the chloroplast genome, and the transfer of cpDNA to
the nucleus was detected by screening for kanamycin-resistant
seedlings in progeny. A screen for kanamycin-resistant seedlings
was conducted with about 250,000 progeny produced by fertili-
zation of wild-type females with pollen from plants containing
cp-neoSTLS2. Sixteen plants of independent origin were identi-
fied and their progenies showed stable inheritance of neoSTLS2,
characteristic of nuclear genes. Thus, we provide a quantitative
estimate of one transposition event in about 16,000 pollen grains
for the frequency of transfer of cpDNA to the nucleus. In addition
to its evident role in organellar evolution, transposition of
cpDNA to the nucleus in tobacco occurs at a rate that must
have significant consequences for existing nuclear genes.
The genomes of cytoplasmic organelles have been depleted
during endosymbiotic evolution, as many genes were either lost
or transferred to the nucleus. The plastome of higher plants has
been reduced to approximately 130 genes from an ancestral cyano-
bacterial genome that probably contained over 3,000 genes
2
.It
has been estimated that 1,700 protein-coding nuclear genes of
Arabidopsis thaliana were acquired from cyanobacteria
1
. Even
greater genetic erosion has occurred during mitochondrial genome
evolution
3
. The rps10 gene, located in the mitochondrial genome in
most angiosperms, has been transferred independently to the
nucleus hundreds of times during endosymbiotic evolution
4
. Simi-
lar multiple independent gene transfers from the chloroplast to the
nuclear genome have also been reported
5
.
Long tracts of extant organelle DNA are also found in the
eukaryotic chromosomes of plants. For example, about 620 kilo-
bases (kb) of mitochondrial DNA (mtDNA) are found on chromo-
some 2 of Arabidopsis
6
, and there are about 33 kb of cpDNA on
chromosome 10L of rice
7
. Indeed, eukaryotes generally contain
DNA sequences in their nuclear genome that show close similarity
Figure 1 Introduction of aadA and neoSTLS2 genes into the tobacco plastid genome by
homologous recombination. a, Construction and integration of pPRV111A::neoSTLS2.
Restriction sites are: A, ApaI; B, BamHI; E, EcoRI; H, HindIII. b, Southern analysis of two
transplastomic lines. Total DNA (5
m
g) was digested with BamHI and hybridized with the
probes indicated (see a). c, Northern analysis of transplastomic lines and their progeny.
Total leaf RNA (3
m
g) was hybridized with the neo probe (see a). 18S RNA stained with
ethidium bromide provides a loading control. Molecular sizes (in kilobases (kb)) are
indicated on the left. n þve, nuclear neoSTLS2 control line.
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to organellar DNA
8–11
, suggesting that transfer of chloroplast and
mtDNA are ongoing, possibly frequent processes.
Transfer of DNA from yeast mitochondria to the nucleus, in the
form of a non-integrating recombinant plasmid, occurs at a rate of
one in 2.5 £10
4
cells per generation
12
, but nothing is known about
the frequency of transposition of cpDNA to the nucleus. To quantify
this process, we inserted a nuclear-specific neoSTLS2 gene (ref. 13)
together with a plastid-selectable aminoglycoside 3 0-adeyltransfer-
ase marker gene, (aadA)
14
, into the plastome of tobacco (Fig. 1a).
The aadA gene confers spectinomycin resistance, enabling selection
of transplastomic cells. The neoSTLS2 gene features a constitutive
plant viral promoter, CaMV-35S
15
, and a nuclear intron. It was
designed to be functional only when transposed to the nucleus
and to confer kanamycin resistance in young seedlings. An intron,
from the potato nuclear ST-LS1 gene
16
, was integrated within the
reading frame to prevent synthesis of a functional protein in the
chloroplast
13
.
Two independent transplastomic (tp) lines (tp7 and tp17),
containing both neoSTLS2 and aadA, were obtained by particle
bombardment of tobacco leaves with pPRV111A::neoSTLS2
(Fig. 1a). DNA blot analysis confirmed the homoplasmic presence
of the transgenes in tp7 and tp17 lines (Fig. 1b). Homoplasmy is not
essential for our transposition experiments, but it may be critical for
genetic stability of the transplastomic lines in the absence of
spectinomycin selection.
Crosses were made to determine the inheritance and function-
ality of the selectable markers in the two transplastomic lines.
Seeds set from tp7 or tp17 as the female parent were able to
germinate on medium containing 500
m
gml
21
spectinomycin,
and develop uniformly into green, healthy seedlings similar to a
positive control, SSuH2 (ref. 17), containing a plastid aadA gene
(Fig. 2a). In contrast, seedlings of wild-type tobacco and seedlings
from wild-type females crossed with tp7 or tp17 as male parents,
bleached rapidly on spectinomycin medium (Fig. 2a and Table 1).
This reciprocal difference in the inheritance of aadA is characteristic
of maternal inheritance of plastid genes in tobacco.
Notably, when the seeds set from tp7 or tp17 female parents were
grown on medium containing 150
m
gml
21
kanamycin, all seedlings
showed partial resistance to the antibiotic. They were uniformly
paler green and smaller in size than positive control seedlings from a
nuclear neo tobacco line, NS23 (ref. 18; Fig. 2b and Table 1). Wild-
type seedlings and seedlings from wild-type females crossed with
tp7 or tp17 male parents were all fully sensitive to kanamycin
Figure 2 Seedling tests for resistance to spectinomycin and kanamycin. a,b, Seedlings
from six progenies grown on medium containing 500
m
gml
21
spectinomycin (a)or
150
m
gml
21
kanamycin (b) with positive (SSuH2 or NS23) and wild-type controls. c, One
kanamycin-resistant plant among the progeny of self-pollinated tp7 plants on kanamycin
(150
m
gml
21
) and geneticin (20
m
gml
21
) medium. d, Segregation of kanamycin
resistance in progeny of wild type £tp-kr1 plants on 150
m
gml
21
kanamycin medium.
e, A kanamycin-resistant plant obtained in the progeny of wild type £tp7 cross.
f, Progenies of four kanamycin-resistant plants with controls on spectinomycin medium.
Table 1 Progenies analysed for resistance to spectinomycin and kanamycin
Cross
Spectinomycin
(500
m
gml
21
)
Kanamycin
(150
m
gml
21
)
kr:ks ratio
rsr s (P)
.............................................................................................................................................................................
Section I
tp7 self 168 0 0 827†
—
tp7 £WT 129 0 0 685†
—
WT £tp7 0 164 7 92,900
—
tp17 self 90 0 0 1,022†
—
tp17 £WT 127 0 0 532†
—
WT £tp17 0 90 11 165,000
—
Section II
WT £tp-kr1 0 142 166 149 1:1 (NS)
kr1 self ND ND 140 34 3:1 (NS)
Section IIIA
kr3 self 0 80 137 42 3:1 (NS)
kr6 self 0 38 107 35 3:1 (NS)
kr7 self 0 49 64 12 3:1 (NS)
kr8 self 0 82 165 49 3:1 (NS)
kr9 self 0 39 51 13 3:1 (NS)
kr10 self 0 35 62 17 3:1 (NS)
WT £kr12 0 79 129 120 1:1 (NS)
WT £kr13 0 100 133 133 1:1 (NS)
kr14 self 0 41 64 18 3:1 (NS)
kr16 self 0 48 126 60 3:1 (NS)
kr17 self 0 64 110 44 3:1 (NS)
kr18 self 0 40 52 15 3:1 (NS)
Section IIIB
kr4 self 0 76 136 330 3:1 (*)
WT £kr4 0 84 80 72 1:1 (NS)
kr5 self ND ND 39 144 3:1 (*)
WT £kr5 0 73 72 76 1:1 (NS)
kr11 self 0 59 12 105 3:1 (*)
kr19 self 0 54 36 59 3:1 (*)
Section IIIC
kr2 self 0 81 0 185 3:1 (*)
WT £kr2 ND ND 0 224 1:1 (*)
kr15 self 0 39 0 162 3:1 (*)
WT £kr15 ND ND 0 311 1:1 (*)
.............................................................................................................................................................................
Progenies are grouped in sections according to the pattern of inheritance. Section III is subdivided to
reflect genotypic and phenotypic differences. The parental origins of the 18 kanamycin-resistant
plants in section III are not specified, as tp7 and tp17 were indistinguishable in every respect (Figs
1c, 2a, b and 3c, d). ND, not determined; NS, not significant; kr, kanamycin resistant; ks, kanamycin
sensitive; r, resistant; s, sensitive; WT, wild type.
*P.0.01 (
x
2
).
†Partial resistance to 150
m
gml
21
kanamycin.
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(Fig. 2b). These results indicate that the partial kanamycin-resistant
phenotype is strictly maternally inherited, as expected for
cp-neoSTLS2 (refs 14, 19), and demonstrate that there is no
functional neoSTLS2 gene in the nucleus of tp7 or tp17 lines,
which could have been introduced concomitantly during chloro-
plast transformation.
Two neo-hybridizing transcripts (3.3 and 2.2 kb) were identified
in tp7 and tp17 RNA that were larger than correctly processed neo
transcripts (1.2 kb) in RNA of a nuclear neoSTLS2 control line,
derived from pCMneoSTLS2 (ref. 13; Fig. 1c). However, a low level
of the intron-spliced messenger RNA was detected in these two
transplastomic lines by the more sensitive polymerase chain reac-
tion with reverse transcription (RT–PCR) (data not shown). There-
fore cp-neoSTLS2 in the chloroplast gives rise to relatively abundant
transcripts that, although inefficiently processed, probably account
for the partial kanamycin resistance observed in the transplastomic
lines and their progenies (Fig. 2b). To suppress this partial resist-
ance, several analogues of kanamycin were tested. Supplementation
of kanamycin medium with a low concentration of geneticin was
found to be effective (Fig. 2c).
One kanamycin-resistant seedling was identified in an initial
screen of 9,300 seeds from the progeny of self-pollinated tp7 on
kanamycin and geneticin medium (Fig. 2c). This plant was desig-
nated tp-kr1 to reflect that it probably contained both chloroplast
and nuclear copies of neoSTLS2. Abundant, correctly processed neo
transcripts (1.2 kb) were identified in tp-kr1 RNA in addition to the
3.3- and 2.2-kb plastid transcripts (Fig. 1c). These 1.2-kb transcripts
remained in kanamycin-resistant progeny after wild-type females
were crossed with tp-kr1, whereas the 3.3- and 2.2-kb chloroplast
transcripts were absent owing to uniparental inheritance (data not
shown).
To gain further evidence that the transferred cpDNA had inte-
grated into the nucleus of tp-kr1, segregation analysis of kanamycin
and spectinomycin resistance was applied to the progeny of wild
type £tp-kr1 crosses. A 1:1 segregation ratio (kanamycin resistant/
kanamycin sensitive) was observed (Fig. 2d and Table 1), demon-
strating that at least one expressed copy of neoSTLS2 had integrated
into the nuclear genome of tp-kr1 at a single locus. Seedlings from
this cross were uniformly sensitive to spectinomycin (Table 1),
confirming non-transmission of the paternal transplastome. The
kanamycin-resistant progeny of the wild type £tp-kr1 cross were
designated kr1 to reflect the replacement of the transplastome with
the wild type plastome. Self-fertilization of kr1 yielded a 3:1
segregation ratio (kanamycin resistant/kanamycin sensitive) in the
progeny, demonstrating stable nuclear transmission of the trans-
posed neoSTLS2 gene (Table 1).A single fragment hybridized to neo
and aadA probes in Southern hybridizations of DraI-digested kr1
DNA, consistent with a single cpDNA insert in nuclear DNA (data
not shown). These data provide genetic and molecular evidence of a
nuclear integrant containing neoSTLS2 in tp-kr1. The unequivocal
verification of nuclear integration provided by sequence analysis of
a transplastomic/nuclear junction in kr1 is described later (see
Fig. 3e).
More kanamycin-resistant individuals arising from independent
transposition events were sought to quantify the frequency of
cpDNA transfer by large-scale screening of seeds derived from
Figure 3 Analysis of kanamycin-resistant plants and three transplastomic/nuclear
junctions. a,NcoI (N) and XbaI (X) transplastome fragments (double-headed arrows).
b,NcoI-digested DNA from 13 kanamycin-resistant plants hybridized with neo.c–d,XbaI-
digested DNA from 17 kanamycin-resistant plants hybridized with neo (c)oraadA (d).
Lanes contained 5
m
g of DNA except for tp7 and tp17 (0.005
m
g). e–g, Unique
transplastomic/nuclear junctions (circle enclosing a cross), PCR primers (arrows),
chloroplast sequences (filled boxes), unknown sequences (open boxes) and nuclear
sequences with database homology (light grey boxes). Templates for PCR from the
progeny of wild type £tp-kr1 (e), self-pollinated kr17 (f) or kr18 (g) are shown. 2ve, no
DNA; þve, iPCR product. Gels were stained with ethidium bromide. Fragment sizes are
indicated in kilobases. kr, kanamycin resistant; ks, kanamycin sensitive.
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wild type £tp7 and wild type £tp17 backcrosses. Eighteen kana-
mycin-resistant plants (kr2–19) were identified among approxi-
mately 250,000 seedlings (Fig. 2e and Table 1). Backcross and self-
pollinated progenies of all 18 kanamycin-resistant plants were
uniformly sensitive to spectinomycin (Fig. 2f and Table 1), con-
firming the absence of pollen transmission of transplastomes from
their paternal transplastomic lines. Kanamycin resistance segregated
in the progenies of 16 kanamycin-resistant plants, 12 of which
showed mendelian inheritance: either 3:1 (kanamycin resistant/
kanamycin sensitive) after self-pollination or 1:1 after crossing to
wild type (Table 1). Therefore at least one expressed copy of
neoSTLS2 is integrated into the nuclear genome at a single locus
in these plants. The other four progenies of self-pollinated plants
(kr4, kr5, kr11 and kr19) had segregation ratios that deviated
significantly from 3:1. However, normal 1:1 ratios were restored
in wild type £kr4 and wild type £kr5, the two backcross progenies
tested (Table 1). Southern analysis identified multiple copies of
neoSTLS2 in these four plants, indicating that complex transgene
interactions
20
may be responsible for the abnormal ratios observed
in the progeny of self-pollinated plants (Fig. 3c).
Progenies of two kanamycin-resistant plants (kr2 and kr15)
derived from self-pollination or backcrossing to wild-type females,
showed uniform sensitivity to kanamycin (Table 1). Southern
analysis confirmed that the neoSTLS2 gene was present in both
parent plants (Fig. 3b) but absent from any offspring (data not
shown), indicating that the nuclear neoSTLS2 gene failed to trans-
mit. These data suggest that deletion may follow insertion at some
chromosomal sites.
Total DNA samples from kr1 and 12 kanamycin-resistant plants
from the large-scale screen were investigated to establish the
independence of the transposition events. NcoI digestion and
hybridization with the neo probe revealed two to four hybridizing
fragments in each sample (Fig. 3a, b). All 13 kanamycin-resistant
plants contained different sized fragments compared with the
control nuclear plant. As neoSTLS2 contains an NcoI site in its
coding region, two neo-hybridizing fragments (3.4 and 5.9 kb) are
expected in the transplastome (Fig. 3a). Ten out of the thirteen plant
DNAs contained these two fragments, as did a low loading of DNA
from tp17 (Fig. 3b). Therefore at least 9.3 kb of DNA, including
aadA, neoSTLS2 and adjacent cpDNA, was transferred from the
transplastome and integrated into their nuclear genomes. These
aadA/neoSTLS2 integrants are flanked by at least 6.5 kb of cpDNA,
which is larger than the plastid targeting sequence (3.0 kb) in the
pPRV111A::neoSTLS2 transformation vector (Fig. 1a). This rules
out the possibility that the neo-hybridizing fragments in most of the
kanamycin-resistant plants could originate from any source other
than the experimental transplastome.
To resolve further the independence of integration events, DNA
samples from all kanamycin-resistant plants, except for kr2 and
kr15, which did not transmit kanamycin resistance, were digested
with XbaI for Southern analysis. The neo probe hybridized to one or
more fragments, which differed in size and/or number, in each
sample (Fig. 3c). Hybridization of the same membrane with the
aadA probe similarly revealed fragments of different size and/or
number in each sample (Fig. 3d). The patterns of hybridizing
fragments for each DNA sample did not resemble that from any
other kanamycin-resistant plant or of the parental transplastomic
lines (Fig. 3c, d), indicating that each plant resulted from an
independent integration event. No hybridization was observed in
pooled DNA from 50 kanamycin-sensitive seedlings derived from
wild type £tp7 or wild type £tp17 crosses (Fig. 3c, d), reconfirm-
ing that no neo or aadA sequences are present in the nuclear genome
of the two transplastomic parental lines.
Junctions between three of the transposed chloroplast sequences
and nuclear DNA were obtained (see Supplementary Information)
by inverse polymerase chain reactions (iPCR). Analysis of a junction
sequence from kr1 (1,315 bp) identified 1,128 bp of non-plastid
DNA adjoining the transposed cpDNA. A sequence located at the 50
end of this non-plastid DNA showed similarity to two nuclear
encoded genes: 67 bp to a Nicotiana plumbaginifolia manganese
superoxide dismutase gene (89%) and 66 bp to tobacco TA-29/
TSJT1 (86%) (EMBL accession numbers Z67979 and X52283,
respectively). No other significant homology was identified within
this sequence, although a 300-bp open reading frame was present at
the 30end. To exclude the possibility that this iPCR product was
artefactual, the junction sequence was amplified by PCR from
genomic DNA. A 0.72-kb junction fragment was amplified from
the parental tp7-kr1 DNA, DNAs from five kanamycin-resistant
progeny of wild type £tp7-kr1 crosses and from the iPCR product
(Fig. 3e). No PCR product was amplified from 5 kanamycin-
sensitive progeny (Fig. 3e) or from the other 16 kanamycin-resistant
plants derived from independent transpositions (data not shown).
These results, together with the genetic and sequence data,
unequivocally demonstrate that the transposed cpDNA in tp-kr1
is integrated into nuclear DNA.
Junction sequences from kr17 (1,451 bp) and kr18 (585bp) DNA
were shown to be unique to the nuclear genome of the plants from
which they were derived and their kanamycin-resistant progeny
(Fig. 3f, g). Sequence analysis of the non-plastid DNA adjacent to
the kr17 and kr18 junctions showed no homology to sequences
deposited in databases except for three short tracts (20–60 bp) that
were similar to chloroplast DNA or mtDNA (Fig. 3f, g). As pre-
existing cpDNA and mtDNA may be very common in the tobacco
genome
11
, it is not surprising to find other organellar DNA in the
vicinity of an experimental integrant. The three junction sequences
indicate that the transposed plastid fragments have integrated into
different nuclear chromosomal locations.
These data demonstrate that DNA is transferred from the
chloroplast and integrated into the nucleus at a frequency of one
in approximately 16,000 tobacco pollen grains (16 independent,
heritable nuclear insertions in 250,000 seedlings). This is a mini-
mum estimate of the proportion of pollen grains that contain newly
transposed cpDNA because our experimental approach would have
identified only those integrants that were sufficiently large to
contain an expressed neoSTLS2 gene. In addition, as the integrants
that we identified are smaller than the entire plastome, we expect
similar transposition events involving other parts of the chloroplast
genome that cannot be monitored by the neoSTLS2 reporter gene.
Transfer of organellar DNA to the nucleus has been a major
driving force in the evolution of eukaryotic cells. Transfer of cpDNA
provides primary sequences for the evolution of new nuclear genes
1
and its frequency is such that integration could have a potentially
wide impact on the function of native nuclear genes and genome
organization. Despite this frequent transfer process the tobacco
nuclear genome does not seem to be continuously expanding, so we
assume that an equilibrium exists between integration and deletion.
It is noteworthy that two kanamycin-resistant plants did not
transmit the transposed plastid sequence to their progeny.
Transfer of cpDNA is the first evolutionary event necessary for
functional establishment of a plastid gene in the nucleus. As the
experimental cp-neoSTLS2 gene contains a constitutive nuclear
promoter, it allowed us to measure the primary rate of transposition
to the nucleus, which does not reflect the problems faced by native
chloroplast genes. The latter need to obtain appropriate nuclear
sequences for transcription, translation and protein transport back
into the chloroplast. A rate several orders of magnitude lower is
expected for such complex DNA arrangements
21
.
The chloroplast is an increasingly attractive location for high level
expression of prokaryote and eukaryote genes
22,23
, and there is the
advantage of natural biological containment afforded by maternal
inheritance that prevents pollen-mediated transmission of the
transplastome in many species
19,24
. Our findings identify a potential
pathway for transfer of plastid transgene DNA in tobacco. However,
genes tailored for expression in the chloroplast are unlikely to
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function in the nucleus as is demonstrated by the absence of
spectinomycin resistance, despite the presence of a nuclear aadA
gene in all of the kanamycin-resistant plants selected. A
Methods
pPRV111A::neoSTLS2 construction and transplastomic plants
pPRV111A::neoSTLS2 was produced by ligating the HindIII fragment containing
neoSTLS2 from plasmid pCMneoSTLS2 (ref. 13) into pPRV111A
14
. This transformation
vector was introduced into the plastids of Nicotiana tabacum L. Petit Havana (N,N) by
biolistic bombardment, and homoplasmic plants were regenerated as described
previously
14
. The homoplasmic plants were transferred to soil and grown in a controlled
environment chambe r with a 14 h light/10 h dark and 25 8C day/20 8C night growth
regime. The photon flux density was approximately 300
m
mol m
22
s
21
at the plant surface.
The nuclear neoSTLS2 control line was generated by biolistic bombardment with plasmid
pCMneoSTLS2 (ref. 13).
Seedling tests for resistance to kanamycin and spectinomycin
Surface-sterilized seeds were plated on 150-mm plates containing 50 ml of 0.5 MS salt
medium
25
and either spectinomycin dihydrochloride (500
m
gml
21
) or kanamycin
sulphate (150
m
gml
21
). For screening seeds of self-pollinated tp7, the latter medium was
supplemented with geneticin disulphate (20
m
gml
21
) when seedlings were two weeks old.
The plates were placed at 25 8C with continuous fluorescent light. To determine
nondestructively the kanamycin-resistance phenotype for verification of transplastomic/
nuclear junctions, leaf pieces were taken from individual seedlings grown in 0.5 MS
medium without kanamycin, and then cultured in MS medium containing 150
m
gml
21
kanamycin and the required plant hormones
25
. Kanamycin resistance was judged by callus
growth.
Molecular analysis
DNA and RNA blot analyses were carried out as described
11,26
. Junction sequences were
obtained using iPCR as described (see http://arabi4.agr.hokudai.ac.jp/ArabiE/protocols/
general/general.html). Key differences in the restriction fragment sizes between nuclear
and transplastomic DNA were used to design cpDNA primers adjacent to the site of
integration. These primers were used in conjunction with a second primer specific for
either aadA or neoSTLS2. Primers used in iPCR were 5 0-GAAGTTTCCAAAAGGTCGTT-
30with 5 0-CTCGCCATCTATTTTCATTG-30for kr1, 5 0-CCAGATTCCAAATGAACAAA-
30(f2) with 5 0-CAATAGCCCTCTGGTCTTCT-30(r3) for kr17, and f2 with r3 for kr18.
PCR amplification
11
of the junction sequences was undertaken with primers 5 0-
GCACTGTGTCATTCAATACT-30(f1) and 5 0-CCAATTGTGACATCCCTTCT-30(r1) for
kr1, f2 and 50-GGTTTTCCAAAGGGGTTTT-30(r2) for kr17, and 5 0-GCCGTCATCAC
TAACCATT-3 0(f3) and r3 for kr18.
Received 26 November 2002; accepted 13 January 2003; doi:10.1038/nature01435.
Published online 5 February 2003.
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Supplementary Information accompanies the paper on Nature’s website
(çhttp://www.nature.com/nature).
Acknowledgements We thank the Australian Research Council for financial support; P. Maliga
for pPRV111A; S. Whitney and J.Andrews for facilities and advice on chloroplast transformation,
for SSuH2 seeds and for comments on the manuscript; T. J. Higgins, P. Whitfeld, W. Martin and
R. Lockington for discussion; C. Maas for pCMneoSTLS2; N. Smith for the NS23 seeds; and S. Elts
for technical assistance.
Competing interests statement The authors declare that they have no competing financial
interests.
Correspondence and requests for materials should be addressed to J.N.T.
(e-mail: jeremy.timmis@adelaide.edu.au). Nucleotide sequences of three transplastomic/nuclear
junctions are deposited in EMBL under accession numbers AJ495859 for kr1, AJ517467 for kr17
and AJ517468 for kr18.
..............................................................
Optimal transsaccadic integration
explains distorted spatial perception
Matthias Niemeier*†‡, J. Douglas Crawford†‡ & Douglas B. Tweed*†‡
*Departments of Physiology and Medicine, University of Toronto, 1 King’s College
Circle, Toronto M5S 1A8, Canada
†Canadian Institutes of Health Research, Group for Action and Perception
‡Centre for Vision Research, York University, 4700 Keele Street, Toronto M3J 1P3,
Canada
.............................................................................................................................................................................
We scan our surroundings with quick eye movements called
saccades, and from the resulting sequence of images we build a
unified percept by a process known as transsaccadic integration.
This integration is often said to be flawed, because around the
time of saccades, our perception is distorted
1–6
and we show
saccadic suppression of displacement (SSD): we fail to notice if
objects change location during the eye movement
7,8
. Here we
show that transsaccadic integration works by optimal inference.
We simulated a visuomotor system with realistic saccades, retinal
acuity, motion detectors and eye-position sense, and pro-
grammed it to make optimal use of these imperfect data when
interpreting scenes. This optimized model showed human-like
SSD and distortions of spatial perception. It made new predic-
tions, including tight correlations between perception and motor
action (for example, more SSD in people with less-precise eye
control) and a graded contraction of perceived jumps; we verified
these predictions experimentally. Our results suggest that the
brain constructs its evolving picture of the world by optimally
integrating each new piece of sensory or motor information.
letters to nature
NATURE| VOL 422 | 6 MARCH 2003 | www.nature.com/nature76 © 2003 Nature Publishing Group