The Plant Cell, Vol. 18, 515517, March 2006, www.plantcell.

org 2006 American Society of Plant Biologists

IN THIS ISSUE

Cytoplasmic Male Sterility and Fertility Restoration

Cytoplasmic male sterility (CMS), a condition under which a plant is unable to

produce functional pollen, is widespread
among higher plants. CMS systems represent a valuable tool in the production of
hybrid seed in self-pollinating crop species,
including maize, rice, cotton, and a number
of vegetable crops. Hybrids often exhibit
heterosis, more commonly known as hybrid vigor, whereby hybrid progeny exhibit
superior growth characteristics relative to
either of the parental lines. CMS systems
can be of considerable value in facilitating
efficient hybrid seed production.
There is growing interest in improving
hybrid technology both to help supply food
for the worlds increasing population and to
contribute to land conservation efforts. For
example, the use of hybrid rice enabled
China to reduce the total amount of land
planted to rice from 36.5 Mha in 1975 to
30.5 Mha in 2000 while at the same time
increasing total production from 128 to 189
million tons, representing a yield increase
of 3.5 to 6.2 tons/ha (http://www.fao.org/
rice2004). Understanding the molecular basis
of CMS, as well as other hybrid production
methods involving self-incompatibility and
apomixis, is critical for continued improvements in hybrid technology.
CMS is a maternally inherited trait that is
often associated with unusual open reading
frames (ORFs) found in mitochondrial genomes (Chase and Babay-Laughnan, 2004;
Hanson and Bentolila, 2004). In many
cases, it has been found that male fertility
can be restored by nuclear-encoded fertility
restorer (Rf) gene(s). CMS/Rf systems therefore are also of value in the study of interactions between nuclear and mitochondrial
genomes. On the one hand, sterility results
from mitochondrial genes causing cytoplasmic dysfunction, and on the other, fertility restoration relies on nuclear genes that
suppress cytoplasmic dysfunction.
CMS can arise spontaneously in breeding lines, as a result of wide crosses or the
interspecific exchange of nuclear and

cytoplasmic genomes, or following mutagenesis (Hanson and Bentolila, 2004). For

example, CMS-WA (wild abortive) rice was
developed in indica rice cultivars from
a male-sterile plant found in a natural population of the wild rice Oryza rufipogon
Griff. CMS-Boro II rice arose from a wide
cross based on the cytoplasm of Chinsurah
Boro II (O. sativa subsp indica) and the
nucleus of Taichung 65 (subsp japonica).
The well-known male-sterile Texas cytoplasm in maize arose spontaneously in
a breeding line, and CMS-PET1 cytoplasm
of sunflower arose from an interspecific
cross between Helianthus petiolaris and
H. annuus.
There are a number of different types of
CMS systems with distinct genetic features, both within and among different
species, but key features that appear to
be shared across different types are (1)
CMS is associated with chimeric mitochondrial ORFs, and (2) fertility restoration is often
associated with genes encoding pentatricopeptide repeat (PPR) proteins (Chase
and Babay-Laughnan, 2004; Hanson and
Bentolila, 2004). In this issue of The Plant
Cell, Wang et al. (pages 676687) describe details of the molecular basis of
CMS and fertility restoration in the CMSBoro II system in rice, which are likely to
have far-reaching implications for CMS
systems in general. First, the authors show
that the mitochondrial orf79 associated
with CMS-Boro II encodes a cytotoxic peptide responsible for CMS, and second, they
show that two PPR proteins encoded by
the Rf-1 locus in the Boro II system block
the production of this cytotoxic peptide by
distinct mechanisms (endonucleolytic cleavage and degradation of the dicistronic
mRNA).
In the early 1990s, several groups reported that rice CMS-Boro II is associated
with an abnormal copy of the mitochondrial
gene apt6 (Kadowaki et al., 1990; Iwabuchi
et al., 1993) that produces aberrant mRNA
transcripts containing an additional ORF

named orf79 (Akagi et al., 1994). Interestingly, in a number of well-characterized

systems, CMS is associated with alterations in promoter regions and portions of
coding regions of mitochondrial ATP synthase subunit genes, which raises the
possibility that impaired ATP synthase
activity could be a causal factor in the

Pollen Grains of a Normal Fertile Rice Line and

orf79-Transgenic Rice Plants.
Top, normal fertile rice line; middle, orf79-transgenic rice plants. Fertile pollen grains are darkly
stained, and sterile grains are lightly stained.
Bars 50 mm. Bottom panel shows cosegregation of the orf79 transgene and male sterility (s)
in transgenic plants. f, male-fertile.

516

The Plant Cell

IN THIS ISSUE

disrupted pollen development in CMS lines

in a number of species (Hanson and
Bentolila, 2004). It is more often considered
that transcription of unusual or aberrant
ORFs is causally related to CMS, and it has
been shown that CMS-associated mitochondrial ORFs encode proteins with cytotoxic properties in sunflower (Nakai et al.,
1995) and Brassicaceae species (Duroc
et al., 2005). However, it was unknown if
CMS-Boro II resulted from incorrect translation of atp6 or from translation of downstream sequences including orf79. Akagi
et al. (1994) found that orf79 encodes
a predicted transmembrane protein with
a novel C-terminal region and an N terminus showing similarity to the rice mitochondrial cytochrome oxidase subunit I. It
was suspected that orf79 causes CMS in
Boro II cytoplasm, but definitive proof has
been lacking.
Wang et al. examined the role of orf79 in
CMS, first by testing for possible cytotoxicity of the ORF79 peptide in Escherichia
coli. Expression of the protein was found to
be lethal to the host E. coli cells, with cell
lysis leading to a rapid decrease in cell
density and lethality depending on the
presence of a fiveamino acid segment of
the C-terminal region. The authors next
tested whether ORF79 causes male sterility
by transforming a normal fertile rice line
with orf79 carrying a mitochondrion-transit
signal under the control of the cauliflower
mosaic virus 35S promoter. The transgenic
plants exhibited semi-male-sterility wherein
50% or more of the pollen grains were
aborted (see figure). Semi-male-sterility was
observed because the transgene is present
in only a portion of pollen grains after meiosis.
Female fertility was unaffected in the transgenic lines, and the semi-male-sterility
phenotype of the T1 progeny cosegregated
with the presence of the transgene with
a 1:1 segregation ratio, indicating that the
orf79 transgene was transmitted normally
through the female germline but poorly or
not all through pollen. These results show
that orf79 encodes a cytotoxic peptide that
causes CMS in rice.
In addition, using immunoblot analysis,
Wang et al. show that, despite the constitutive RNA expression of the gene, ORF79
protein accumulates specifically in the

microspores of a CMS line but is absent

in sporophytic tissues (as well as the
microspores of fertility-restored plants).
They propose that there may be a posttranslational regulatory mechanism that
suppresses the accumulation of the protein, which could explain the genetic
feature of gametophytic male sterility and
why orf79 does not disrupt the development of sporophytic tissues.
Wang et al. then sought to characterize
the nature of the complex Rf1 chromosomal region and clarify the molecular
mechanism underlying fertility restoration.
The restoring allele Rf-1 is present in some
indica rice lines, whereas most lines of the
subspecies japonica carry a nonrestoring
rf-1 allele. Previous research had shown
that Rf-1 encodes a PPR protein that functions in fertility restoration of CMS-Boro II
(Kazama and Toriyama, 2003; Akagi et al.,
2004; Komori et al., 2004). Akagi et al.
(2004) showed that Rf-1 is a complex locus
containing multiple copies of genes encoding PPR proteins. One of the genes, Rf-1A,
encoded a predicted PPR protein of 791
amino acids that contained a mitochondriatargeting signal and cosegregated with fertility restoration. This was the same gene
cloned by Kazama and Toriyama (2003)
(called PPR8-1) and Komori et al. (2004)
(called PPR791) and thus has been considered to be the single gene responsible
for fertility restoration. Akagi et al. (2004)
also identified two other genes thought
to be nonfunctional in fertility restoration:
Rf-1B, which encoded a truncated protein
that lacked a mitochondrial-transit signal,
and Rf-1C, which encoded a protein of high
similarity to Rf-1A but was outside of
the crossover point of the experimental
recombinants.
Wang et al. used map-based cloning to
sequence a 37-kb region surrounding Rf-1.
They identified two genes that encode PPR
proteins, called Rf1a and Rf1b, which were
found by complementation testing to function in fertility restoration in this system.
Rf1a corresponds to Rf1A/PPR8-1/PPR791
identified in previous studies, whereas
Rf1b is another gene at this locus that has
not been described previously. Wang et al.
show that the RF1A and RF1B proteins
both function to restore male fertility by

blocking ORF79 production via somewhat

different mechanisms. RF1A is shown to
mediate endonucleolytic cleavage of the
dicistronic atp6/orf79 mRNA at three major
regions, each with multiple cleaving sites,
whereas RF1B mediates degradation of
atp6/orf79 mRNA with no detectable intermediates. RF1A function was found to be
epistatic over RF1B, such that when both
were present, atp6/orf79 mRNA was preferentially cleaved by RF1A, and the cleavage products were not susceptible to
further degradation mediated by RF1B.
The authors suggest that different fertility
restorer lines may carry either or both
functional copies of Rf1a and Rf1b, and
the restorer lines in previous studies presumably did not carry a functional Rf1b
allele.
PPR proteins constitute a large family,
with .400 members in Arabidopsis and
rice that are thought to be RNA binding
proteins involved in posttranscriptional
processes (RNA processing and translation) in mitochondria and chloroplasts, but
little data exist on the functions of individual
proteins in this family (Lurin et al., 2004).
Lurin et al. (2004) hypothesized that they
function as sequence-specific adaptors for
a variety of other RNA-associated proteins.
This idea was supported by SchmitzLinneweber et al. (2005), who showed that
the maize PPR protein CRP1 influences
expression of chloroplast genes through
association with specific mRNAs, and
Kotera et al. (2005), who showed that
PPR proteins are involved in mRNA editing
in chloroplasts. Bentolila et al. (2002) suggested an mRNA processing function for Rf
PPR function after identifying the first Rf
gene in petunia, which was found to encode the PPR protein Rf-PPR592. They
observed that the presence of the restorer
Rf-PPR592 led to a decrease in the gene
product of the aberrant mitochondrial ORF
pcf and concluded that Rf-PPR592 is likely
involved in mediating a reduction in mRNA
accumulation. Wang et al. provide definitive support for this hypothesis, showing
that Rf PPR proteins participate both in
endonucleolytic cleavage (RF1A) and degradation (RF1B) of specific mRNAs. They
further show that RF1A functions to promote the editing of normal atp6 mRNA

March 2006

517

IN THIS ISSUE

independently of its cleavage of dicistronic

atp6/orf79 mRNA and role in fertility restoration and suggest that this may be its
primary function.
The results of Wang et al. have important
implications for other CMS systems. For
example, the A3 CMS system in sorghum
has similar features with the CMS-Boro II
system in rice: the mitochondrial CMS gene
of sorghum, orf107, is similar to rice orf79
(Tang et al., 1996), and fertility restoration in
sorghum is associated with the editing
status of mitochondrial atp6 (Pring et al.,
1999). In addition, previous work has
suggested that Rf loci associated with
fertility restoration in the genetically distinct
rice CMS systems CMS-WA (Zhang et al.,
2002) and CMS-HL (Liu et al., 2004) map to
the same PPR gene cluster as the CMSBoro II Rf-1 locus. Wang et al. hypothesize
that a number of different genes within this
PPR cluster have been recruited as fertility
restorers with divergent molecular functions. Analyses of these clusters in other
CMS systems are needed for a complete
understanding of the evolution and molecular basis of CMS.
Nancy A. Eckardt
News and Reviews Editor
neckardt@aspb.org

REFERENCES
Akagi, H., Nakamura, A., Yokozeki-Misono,
Y., Inagaki, A., Takahashi, H., Mori, K., and
Fujimura, T. (2004). Positional cloning of the
rice Rf-1 gene, a restorer of BT-type cytoplasmic male sterility that encodes a mitochondriatargeting PPR protein. Theor. Appl. Genet.
108, 14491457.
Akagi, H., Sakamoto, M., Shinjyo, C., Shimada,
H., and Fujimura, T. (1994). A unique se-

quence located downstream from the rice

mitochondrial apt6 may cause male sterility.
Curr. Genet. 25, 5258.
Bentolila, S., Alfonso, A.A., and Hanson, M.R.
(2002). A pentatricopeptide repeat-containing
gene restores fertility to cytoplasmic malesterile plants. Proc. Natl. Acad. Sci. USA 99,
1088710892.
Chase, C., and Babay-Laughnan, S. (2004).
Cytoplasmic male sterility and fertility restoration by nuclear genes. In Molecular Biology
and Biotechnology of Plant Organelles, H.
Daniell and C. Chase, eds (Dordrecht, The
Netherlands: Kluwer Academic Publishers),
pp. 593622.
Duroc, Y., Gaillard, C., Hiard, S., Defrance,
M.-C., Pelletier, G., and Budar, F. (2005).
Biochemical and functional characterization of
ORF138, a mitochondrial protein responsible
for Ogura cytoplasmic male sterility in Brassiceae. Biochimie 87, 10891100.
Hanson, M.R., and Bentolila, S. (2004). Interactions of mitochondrial and nuclear genes
that affect male gametophyte development.
Plant Cell 16, S154S169.
Iwabuchi, M., Kyozuka, J., and Shimamoto, K.
(1993). Processing followed by complete editing of an altered mitochondrial apt6 RNA
restores fertility of cytoplasmic male sterile
rice. EMBO J. 12, 14371446.
Kadowaki, K., Suzaki, T., and Kazama, S.
(1990). A chimeric gene containing the 5#
portion of atp6 is associated with cytoplasmic
male-sterility of rice. Mol. Gen. Genet. 224,
1016.
Kazama, T., and Toriyama, K. (2003). A
pentatricopeptide repeat-containing gene that
promotes the processing of aberrant apt6
RNA of cytoplasmic male-sterile rice. FEBS
Lett. 544, 99102.
Komori, T., Ohta, S., Murai, N., Takakura, Y.,
Kuraya, Y., Suzuki, S., Hiei, Y., Imaseki, H.,
and Nitta, N. (2004). Map-based cloning
of a fertility restorer gene, Rf-1, in rice (Oryza
sativa L.). Plant J. 37, 315325.
Kotera, E., Tasaka, M., and Shikanai, T.
(2005). A pentatricopeptide repeat protein is

essential for RNA editing in chloroplasts.

Nature 433, 326330.
Liu, X.Q., Xu, X., Tan, Y.P., Li, S.Q., Hu, J.,
Huang, J.Y., Yang, D.C., Li, Y.S., and Zhu,
Y.G. (2004). Inheritance and molecular mapping of two fertility-restoring loci for Honglian
gametophytic cytoplasmic male sterility in rice
(Oryza sativa L.). Mol. Genet. Genomics 271,
586594.
Lurin, C., et al. (2004). Genome-wide analysis of
Arabidopsis pentatricopeptide repeat proteins
reveals their essential role in organelle biogenesis. Plant Cell 16, 20892103.
Nakai, S., Noda, D., Kondo, M., and Terachi,
T. (1995). High-level expression of a mitochondrial orf-522 gene from the male-sterile sunflower is lethal to Escherichia coli. Breed. Sci.
45, 233236.
Pring, D.R., Tang, H.V., Howad, W., and
Kempken, F. (1999). A unique two-gene
gametophytic male sterility system in Sorghum involving a possible role of RNA editing
in fertility restoration. J. Hered. 90, 386393.
Schmitz-Linneweber, C., Williams-Carrier, R.,
and Barkan, A. (2005). RNA immunoprecipitation and microarray analysis show a chloroplast pentatricopeptide repeat protein to be
associated with the 5# region of mRNAs
whose translation it activates. Plant Cell 17,
27912804.
Tang, H.V., Pring, D.R., Shaw, L.C., Salazar,
R.A., Muza, F.R., Yan, B., and Schertz, K.F.
(1996). Transcript processing internal to a mitochondrial open reading frame is correlated
with fertility restoration in male-sterile sorghum. Plant J. 10, 123133.
Wang, Z., et al. (2006). Cytoplasmic male
sterility of rice with Boro II cytoplasm is
caused by a cytotoxic peptide and is restored
by two related PPR motif genes via distinct
modes of mRNA silencing. Plant Cell 18,
676687.
Zhang, Q., Liu, Y.G., Zhang, G., and Mei, M.
(2002). Molecular mapping of the fertility
restorer gene Rf4 for WA cytoplasmic male
sterility in rice. Yi Chuan Xue Bao 29, 1001
1004.