Centromere renewal and replacement
in the plant kingdom
R. Kelly Dawe
Departments of Plant Biology and Genetics, University of Georgia, Miller Plant Sciences Building, Athens, GA 30602
C
entromere specification has
been a topic of intense interest
since it became clear that, under
the right conditions, human centromeres
can form over noncentromeric
sites. At least 60 human ``neocentromeres''
have been described that retain no
vestige of the familiar -satellites and that
map to apparently random locations in
the genome (1). At the functional and cell
biological levels (e.g., association with kinetochore
proteins), not a single difference
has been detected between standard
human centromeres and neocentromeres.
These and similar data from Drosophila
suggest that animal centromeres are initiated
in large part by epigenetic mechanisms
(2). In this issue and a recent issue
of PNAS, two new articles (3, 4) extend
these observations to plants. Lee et al. (3)
provide a new evolutionary perspective on
centromere evolution, demonstrating that
satellite repeats are gained and lost at
astonishing rates. Nasuda et al. (4) take
the story a step further to show that barley
centromeres can move to new positions
and that satellite DNA is not necessary
for efficient centromere formation.
An Ancient Centromere Repeat Has
Spawned Multiple Variants in the
Grass Family
In both plants and animals, the major
centromeric DNAs are small 100- to
200-bp satellite repeats that usually are
organized in very long arrays. In rice, the
major repeat is CentO, and in maize it is
CentC. Both are known to interact with
the key centromere protein Centromeric
Histone H3 (CENH3) (5, 6). Grass centromeres
also contain a specialized class
of centromeric retroelements (CR elements)
that bind to CENH3 and are thoroughly
interspersed with the satellites (5).
The presumption is that satellite arrays
are the primary centromere repeats (5­7),
whereas CR elements are either efficient
centromere parasites (8) or facilitate the
establishment of a centromeric state (9).
A central issue in centromere research
is the ``centromere paradox'' (10), the apparent
conflict between the importance of
centromeric DNA and the fact that it
evolves so quickly that identifying conserved
sequences is often impossible. An
answer to the paradox will require, in
part, a better understanding of the variation
that is present within large clades of
related species. Toward this end, Lee et al.
(3) took a biochemical approach to identify
centromere repeats in two understudied
rice species, Oryza rhizomatis and
Oryza brachyantha. There are numerous
allotetraploids in the Oryza genus, and the
species are often referred to by the genomes
they contain (e.g., BB, CC, BBCC,
etc.). As shown in Fig. 1, O. rhizomatis is a
diploid with a CC genome, and O. brachyantha
is a diploid with a FF genome. Both
species had been previously shown to lack
CentO (11). Lee et al. (3) used chromatin
immunoprecipitation to partially purify
centromeric nucleosomes, extracted
the associated DNA, and prepared
small-insert libraries for sequencing. In
O. rhizomatis (CC), the major repeats
turned out to be variants of CentO named
CentO-C1 and CentO-C2. CentO-C2 is
present at centromeres and telomeres,
and CentO-C1 is a centromere-specific
sequence.
CentO-C1 shares sequence homology
over an 80-bp region that also is conserved
between rice CentO and maize
CentC. Phylogenetic analysis suggests that
CentC, CentO, and CentO-C1 diverged at
roughly the same time several million
years before the emergence of Oryza (3)
(Fig. 1). By using pattern-matching
software, Lee et al. also were able to identify
significant homology between the conserved
CentO CentC CentO-C1 domain
and a known centromere repeat in pearl
millet. These data provide strong evidence
for an ancestral centromere repeat and a
conserved sequence motif within it. It is
unclear what function such a motif might
provide. It is possible that the motif represents
a conserved protein-binding site;
however, no sequence-specific centromere
binding proteins have been identified in
plants. A second possibility, preferred by
the authors, is that the motif confers
structural stability to the repeat (3).
Genomewide Centromere Replacement
in a Short Time Frame: Evidence for
Centromere Drive?
A recently proposed model for centromere
evolution (and potential solution to
the centromere paradox) supposes that
centromere repeats can influence how
frequently they are recovered in progeny
and evolve accordingly (12). Under this
view, known as the centromere drive hypothesis,
centromeres that are particularly
good at chromosome segregation will arrive
in reproductive cells more often than
those that are not. Assuming that centromere
function is at least partially determined
by DNA sequence, it stands to
reason that sequences that can confer segregation
advantages will increase in number
(13). It is an appealing idea, but like
many evolutionary models it is difficult to
test. For instance, the prevalence of
CentO-C1 in O. rhizomatis suggests that it
may have had a segregation advantage
over other repeats in this lineage. However,
whether it was sequence-based adaptive
evolution or genetic drift is difficult to
determine because both CentO and
CentO-C1 preexisted in the progenitor
species. More to the point would be a new
centromere repeat that arises in a single
species and sweeps through the genome.
As a part of their chromatin immunoprecipitation
cloning study, Lee et al. (3)
cloned centromeric DNA from an African
rice species known as O. brachyantha
(FF). Surprisingly, none of the 96 repeats
sequenced proved to be CentO, Cento-C1,
or CentO-C2. Instead, they found
CentO-F, which lacks any detectable homology
to other centromere repeats.
See companion articles on pages 11793 in this issue and
9842 in issue 28 of volume 102.
E-mail: kelly@plantbio.uga.edu.
 2005 by The National Academy of Sciences of the USA
Fig. 1. The distribution of centromere repeats in
several rice genomes as described by Lee et al. (3).
The phylogeny provided by Ge et al. (18) was used
as a template to show evolutionary relationships.
Cultivated rice (O. sativa) contains the AA genome,
O. rhizomatis contains the CC genome, and O.
brachyantha contains the FF genome. The major
repeats are indicated with symbols; the size of
symbol provides a rough indicator of how prevalent
the repeat is. Major events in centromere evolution
are indicated with red bars.
www.pnas.org cgi doi 10.1073 pnas.0505100102 PNAS August 16, 2005 vol. 102 no. 33 11573­11574
COMMENTARY
CentO-F is found at every centromere in
O. brachyantha and has a copy number
that is similar to that of CentO in cultivated
rice ( 20,000 per haploid genome).
It is possible that CentO-F existed in the
Oryza progenitor(s), but it seems unlikely,
because there was no evidence of the repeat
in any of the other 16 species
analyzed.
Another remarkable characteristic of
O. brachyantha (FF) is that it contains few
if any canonical CR elements (known as
CRR in rice). CR elements are the most
conserved and reliable markers of cereal
centromeres and interact with CENH3 as
efficiently as satellite DNA (5, 6).
CentO-F may have usurped the centromere
functions that CR elements normally
provide (9) or may simply be
spreading so fast that the CR elements
cannot keep pace (3). It is difficult to estimate
how quickly it occurred, but a reasonable
guess would be that CentO-F
evolved and drove out CentO, CentO-C1,
and CRR in a period of 7 million to 9
million years (approximate age of the FF
genome; E. Kellogg, personal communication).
By comparison, the -satellites of
New and Old World monkeys retain
64% sequence homology, even though
the two superfamilies diverged 35 million
years ago (13, 14). With respect to
rapid evolution and shear aggressiveness,
the behavior of rice CentO-F is consistent
with the centromere drive model (3, 12).
Plant Centromeres Can Function Without
Canonical Satellite Repeats
The concept of the epigenetically
determined centromere became firmly
entrenched when the first human neocentromere
was identified and carefully documented
(1, 2). Facultative neocentromeres
also had been identified in maize and rye,
but unlike animal neocentromeres, classic
plant neocentromeres are meiosis-specific
and lack kinetochores (15). Thus, until the
work of Nasuda et al. (4), it remained possible
that fully functional neocentromeres
and the implied centromere plasticity
were unique to animals.
Several ``gametocidaľ' chromosomes
from Aegilops (a wheat relative) have the
unique property of inducing breakage on
other chromosomes when introduced into
wheat. When chromosomes from barley
are introduced into such a wheat­Aegilops
addition line, they too are subject to
breakage. It was in such a gametocidal
background that a series of barley translocation
and deletion chromosomes were
identified (Fig. 2). The two chromosomes
analyzed in the most detail were telosomes
containing a single arm of barley
chromosome 7H. One of the telosomes
had lost the original centromere (7HS*)
and another had lost the centromere and
at least a portion of the flanking pericentromeric
DNA (7HS**). Importantly, all
known centromere repeats and CR elements
were absent on 7HS* and 7HS**.
Nevertheless, both truncated chromosomes
were completely stable in genetic
crosses, and 7HS* reacted strongly with
antisera to at least four known kinetochore
proteins.
The data are very similar to prior
observations in Drosophila that suggest
centromeres can spread into flanking
chromatin (16). The primary difference is
that the Drosophila centromere moved
only into euchromatin, whereas barley
centromere 7H appeared to move into (or
over) heterochromatin. It is possible that
in both cases the neocentromeres were
close enough to an established centromere
that small amounts of CENH3 and associated
proteins were carried over (4, 16).
The centromeric chromatin may then have
spread and adopted a domain similar in
size to the original centromere but with
few or none of the original DNA
sequences.
Nasuda et al. (4) have demonstrated
convincingly that plant centromeres do
not require satellite repeats to function
normally and provided new support for
the view that neocentromeres are established
epigenetically. Indeed, although
tandem repeat arrays can initiate new
centromeres under experimental conditions
(17), there is little evidence that it
occurs naturally. It appears that centromere
repeats normally accumulate after a
new centromere has been established, selfishly
and (at least initially) without regard
to organismal fitness (6, 10, 12, 15).
Despite this unlikely mode of evolution,
simple repeats have emerged as dominant
centromeric sequences in nearly all plants
and animals. Future work is likely to focus
on the proteins involved in the earliest
stages of (neo)centromere formation
as well as these still-enigmatic repeats,
including what sequence features allow
them to contribute to centromere formation
and what proteins facilitate their
interactions with the kinetochore.
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Fig. 2. The origin of barley neocentromeres as described by Nasuda et al. (4). The study began with a
translocation between barley chromosome 7HS and an unidentified wheat chromosome. An isochromosome
derivative of the translocation was identified that had lost the wheat half of the chromosome as well as any
evidence of the barley centromere. From the isochromosome, two telosomic derivatives were produced that
appeared to have lost even larger segments of the original centromere. The telosomes lacked known
centromere repeats from either barley or wheat yet were mitotically and meiotically transmissible.
11574 www.pnas.org cgi doi 10.1073 pnas.0505100102 Dawe