The classical Ubisch bodies carry a sporophytically
produced structural protein (RAFTIN) that is
essential for pollen development
Aiming Wang*, Qun Xia, Wenshuang Xie†, Raju Datla, and Gopalan Selvaraj‡
Plant Biotechnology Institute, National Research Council of Canada, 110 Gymnasium Place, Saskatoon, Saskatchewan, Canada S7N 0W9
Edited by Maarten Koornneef, Wageningen University and Research Centre, Wageningen, The Netherlands, and approved September 11, 2003
(received for review March 4, 2003)
Pollen fecundity is crucial to crop productivity and also to biodi-
versity in general. Pollen development is supported by the tape-
tum, a metabolically active sporophytic nurse layer that devotes
itself to this process. The tapetum in cereals and a vast majority of
other plants is of the nonamoeboid type. Unable to reach out to
microspores, it secretes nutrients into the anther locule where the
microspores reside and develop. Orbicules (Ubisch bodies), studied
in various plants since their discovery ?140 years ago, are a
hallmark of the secretory tapetum. Their significance to tapetal or
pollen development has not been established. We have identified
in wheat and rice an anther-specific single-copy gene (per haploid
nearly eliminated the seed set. The flowers in the transgenics were
normal for female functions, but the pollen collapsed and became
less viable. Further characterization of the gene product, named
and it is accumulated in Ubisch bodies. Furthermore, it is targeted
to microspore exine. Although the carboxyl portion of RAFTINs
shares short, dispersed amino acid sequences (BURP domain) in
the occurrence RAFTIN per se is limited to cereals; neither the
Arabidopsis genome nor the vast collection of ESTs suggests any
obvious dicot homologs. Furthermore, our results show that RAF-
TIN is essential for the late phase of pollen development in cereals.
understood factors that affect grain yield in these highly inbreed-
ing species. Pollen production is adversely affected when the
temperature is too warm or too cold. The molecular aspects of
pollen development in these cereals are still sketchy. Much of the
current information is from other plant systems (1–8). Pollen
grains originate from the innermost layer of the anther, and their
development is sustained by the tapetum, a metabolically active
sporophytic cell layer that surrounds the sporogenous cells. The
6, 7, 9–12), and it is essential for pollen development as shown
from male sterility caused by precocious ablation of the tapetum
(10, 11). There are two major types of tapetum, and their
taxonomic distribution is almost mutually exclusive (13, 14). The
secretory tapetal cell layer is more prevalent and is considered
primitive in contrast to the amoeboid type that reaches out to the
microspores in the anther locule for presumed direct delivery of
the tapetal contents. Wheat and rice have secretory tapetum
(15). The presence of spheroid structures of ?1 ?m is a hallmark
of secretory tapetum in a vast majority of plants. Discovered by
bodies (15). Despite their long history, a definitive function for
Ubisch bodies has not been established. Some consider them to
be no more than a by-product of tapetal metabolism, whereas
others have suggested functions such as transport of sporopol-
lenin, a complex of fatty acid derivatives and phenylpropanoids
that form an extremely inert biopolymer in the exine to resist
physical, biological and chemical attacks (15–17). They arise as
ice and wheat are the staple food for much of the global
population. Pollen fertility is one of the limiting and poorly
pro-orbicules in the tapetum and are extruded to the locular side
where they acquire a sporopollenin coat and remain attached to
the peritapetal wall (17).
Ubisch bodies have not been isolated, likely because of their
small size and physical association to the tapetal cell wall, and
their biochemical characterization has therefore been hindered.
They are absent in Cruciferae members such as Brassica spp. and
Arabidopsis, where the biochemistry and genetics of tapetal
contributions to pollen development have been investigated in
function of Ubisch bodies. Here, we demonstrate that they carry
a sporophytic protein that is targeted to the microspore exine
and that it is essential for pollen development.
Materials and Methods
Plant Materials. Hexaploid spring wheat (Triticum aestivum L. cv.
AC Karma, AABBDD), tetraploid wheat (Triticum turgidum L.
cv. Sceptre, AABB), two diploid wheat species (Triticum urartu,
ssp. nigrum, AA; Triticum tauschii L. china, DD) (22) and rice
(Oryza sativa L. japonica var. Nipponbare; a gift of T. Sasaki,
National Institute of Agrobiological Resources, Ibaraki, Japan)
Cloning, Sequence Analysis, and Nucleic Acid Hybridization. The 5?
sequence of an anther-specific cDNA (A71) identified from an
anther cDNA library of hexaploid wheat (22) was found not to
show any resemblance to the Arabidopsis genome in the nucle-
otide or deduced amino acid sequence (www.arabidopsis.org;
BLAST). A 346-bp amplicon from the 5? portion, PCR-amplified
with primers OL3044 and OL3045, was32P-labeled and used to
information on the PNAS web site). The inserts from all 26
positive clones were sequenced and found to be only of two
polymorphic types. The longest clone of each class, confirmed by
5? RACE as full-length cDNAs, is referred to as taRAFTIN1a
and taRAFTIN1b, respectively. BLAST analysis of the entire
GenBank records identified a rice ortholog (GenBank accession
no. AP000364). The predicted ORF (osRAFTIN1) was RT-PCR
amplified with primers OL4382 and OL4383 and directionally
cloned into the KpnI–XbaI sites of plasmid pBluescript SK
(Stratagene). The genomic counterparts of the wheat and rice
RAFTIN cDNAs, obtained by PCR, were cloned into T?A vector
(Invitrogen) and sequenced. The oligonucleotide synthesis,
This paper was submitted directly (Track II) to the PNAS office.
database (accession nos. AJ575662–AJ575668).
*Present address: Southern Crop Protection and Food Research Centre, Agriculture and
Agri-Food Canada, 1391 Sandford Street, London, ON, Canada N5V 4T3.
†Present address: Department of Chemical Engineering, University of California, Berkeley,
‡To whom correspondence should be addressed. E-mail: firstname.lastname@example.org.
November 25, 2003 ?
vol. 100 ?
no. 24 ?
DNA sequencing and sequences analyses were carried out as
described (22). Genomic DNA isolation and Southern blot
analysis were performed as described (22). The entire ORFs of
taRAFTIN1a and osRAFTIN1 retrieved by PCR were used as
probes for hybridization of genomic DNA. The upstream
genomic region of the taRAFTIN1a and taRAFTIN1b ORFs
were isolated by using a Universal GenomeWalker kit (Clon-
tech). Two nested primers, OL3070 and OL3071, were used for
the first and second PCR. The resulting two fragments of 1.7 kb
for taRAFTIN1a and 2.1 kb for taRAFTIN1b were cloned into a
T?A vector (Invitrogen). A 1,458-bp segment upstream of the
osRAFTIN1 ORF was retrieved by using primers OL3079 and
Plant Vectors and Genetic Transformation. Rice transformation was
as described (23) with plasmid constructs shown in Fig. 7, which
is published as supporting information on the PNAS web site.
generated in a 20-?l reaction containing 5 ?g of total RNA
isolated from appropriate wheat?rice tissues, 0.5 ?g of oli-
go(dT)18and 20 units of SUPERSCRIPT II RNase H?Reverse
Transcriptase (Invitrogen). A total of 150 ng of RNA-derived
cDNA were used for a 100-?l PCR in the presence of 10 units
of TaqDNA polymerase (Amersham Pharmacia). Primers
OL3044 and OL3073 were used for wheat taRAFTIN1a to
produce an 875-bp amplicon, and primers OL3148 and OL3815
were used for a 441-bp amplicon from rice osRAFTIN1. Primers
OL4556 and OL4557 were used for generating 562-bp control
for a housekeeping gene, GAPDH (GenBank accession no.
U31676). All PCRs were carried out with a Techne Genius
thermocycler (Duxford, Cambridge, U.K.): 35 cycles of 94°C, 30
sec; 56°C, 30 sec; and 72°C, 1 min; finally a 10-min extension at
72°C. Five microliters of the reaction was used for agarose gel
Protein Methods. taRAFTIN1a ORF was amplified with primers
OL3174 and OL3175 and cloned in-frame into the BamH1–
EcoRI sites of plasmid pTrxFus (Invitrogen) to yield plasmid
pAMWthio-A71. The fusion protein produced in Escherichia
coli strain GI724 (Invitrogen) was purified and used for immu-
nizing rabbits as described (22). Antiserum IgG was initially
purified (24) and further purified with Affi-Gel 10 (Bio-Rad)
following the suppliers’ instructions. Plant protein extraction
and Western blot analysis were performed essentially as de-
Microscopy. For transmission electron microscopy, samples were
fixed in 3% glutaraldehyde in 0.025 M phosphate buffer (pH 6.8)
overnight at 4°C and postfixed in 1% osmium tetroxide on ice for
8 h. After dehydration in a graded ethanol series, the samples
were embedded in acrylic resin (London Resin, Reading, Berk-
shire, U.K.). Ultra-thin sections (50–70 nm) were made by using
a Reichert Jung Ultracut E microtome (Leica, Vienna, Austria),
and double-stained with 2% (wt?vol) uranyl acetate and 2.6%
(wt?vol) lead citrate. The section was viewed and photographed
with a Philips CM-10 transmission electron microscope (Philips
Electron Optics). In situ RNA hybridization and immunoblotting
were as described (22). Scanning electron microscopy was as
described (26). For immunogold labeling, wheat anthers were
pH 6.8) for 1 h, then with 3% glutaraldehyde in PB for 3 h,
followed by a rinse with PB at 4°C overnight and dehydration in
a graded ethanol series. The anthers were infiltrated with
LR-white resin (London Resin) and polymerized by UV light.
and incubated with blocking solution containing 1% BSA in PBS
buffer (10.14 mM Na2HPO4?1.76 mM KH2PO4, pH 7.4?136.9
mM NaCl?2.69 mM KCl) for 30 min, followed with 1 h of
incubation with the Affi-Gel column-purified antibody in the
blocking solution. After washing with PBS buffer, the sections
were treated with colloidal gold-conjugated anti-rabbit IgG
developed in goat (1:75; EMGAR15; British BioCell Interna-
tional, Cardiff, Wales) for 1 h. After washing with PBS and
acetate for 20 min, washed with distilled water for 1 h, and
incubated with 0.3% lead citrate for 10 min. After a rinse with
distilled water, the sections were viewed and photographed with
a Philips 410 LS electron microscope (Philips Electron Optics).
All of the above steps were performed at room temperature
unless otherwise stated.
RAFTIN Gene of Wheat and Its Paralogs and the Rice Ortholog Are
Highly Anther-Specific in Expression, and RAFTIN Is Essential for Male
Fertility. The expression pattern of RAFTIN1 in wheat and rice
was studied in detail (Fig. 1). The RAFTIN1 transcripts of wheat
and rice (?1.3 kb) were evident in young inflorescence but not
in root, stem, leaf tissues, or emasculated inflorescence. RT-
PCR analyses showed that the negative samples here were
indeed devoid of RAFTIN mRNA. Even after 35 cycles no
amplicon was generated in these, whereas it was found from the
22nd cycle onward in the anther samples. Thus, these experi-
ments established anther-specific expression of RAFTIN1 in
both wheat and rice. Given the spatial specificity in rice and the
absence of an ortholog in the Arabidopsis genome, we generated
RAFTIN-defective phenocopy in rice to determine whether the
gene had a discernible function. Intron hairpin (ihp) RNA-
induced gene silencing strategy (27) was used. A combinatorial
series of eight constructs (Fig. 7) that had rice or wheat ihp
Northern blot analysis of RAFTIN1 expression in hexaploid wheat and rice,
respectively. (A) Probed with the entire taRAFTIN1a ORF. (C) Probed with the
entire osRAFTIN1 ORF. The panels immediately underneath A and C are
ethidium bromide-stained gels. (B and D) RT-PCR analysis of RAFTIN1 expres-
sion in wheat and rice, respectively. For RT-PCR, the panel underneath is
the control RT-PCR of a housekeeping gene, GAPDH (taGAPDH for wheat,
osGAPDH for rice). Primers and PCR conditions were described in Materials
and Methods. Total RNA isolated from different tissues was used for RT-PCR
and Northern blotting analyses. Fl w/o anther, young flower tissue with the
Anther-specific expression of RAFTIN1 in wheat and rice. (A and C)
www.pnas.org?cgi?doi?10.1073?pnas.2231254100Wang et al.
RAFTIN1 sequences were made and introduced into rice by
particle bombardment. Fifty-four transgenic lines with these
constructs and nine lines with a selection marker gene (as a
control) were obtained. RNA isolated from the anthers (at the
vacuolated microspore stage) of a randomly chosen representa-
tive line for each construct was used for Northern blot analysis
to evaluate the silencing efficacy (Fig. 2). Of the eight repre-
sentative ihp transgenic lines, the ones made with pAMW497,
pAMW498, and pAMW506 showed osRAFTIN1 transcript lev-
els of ?5% relative to the control (WT), whereas the others had
10–37.6% (Fig. 2). All of the transgenic lines including those
analyzed above were grown in a greenhouse, and all of them had
a similar vegetative growth phenotype (Fig. 8 A and B, which is
published as supporting information on the PNAS web site).
Tillering and leafing ability, leaf size, internode elongation, and
overall plant sizes were similar, and panicle initiation in all of the
lines occurred ?90–100 days after transplanting. All of the lines
had similar floral morphology, but the spikelet of the osRAF-
TIN1-silenced lines did not open and anthesis did not occur at
maturity even over an extended time (6 weeks) after panicle
emergence. No seeds or only a few seeds per plant (?1 seed per
panicle) were produced in the osRAFTIN1-silenced lines, in
contrast to ?10 seeds per panicle in the control lines (Fig. 8
C–H). The palea in the osRAFTIN1-silenced lines remained
green, whereas that in the control lines turned yellow (Fig. 8 E
and F). The lack of seed set was found to be due to male sterility;
the osRAFTIN1-silenced lines crossed with pollen from a control
line produced seeds, showing that female fertility was unim-
paired (Fig. 8I).
Suppression of RAFTIN1 Impairs Pollen Grain Maturation. The male
sterile phenotype was studied further for investigating the func-
tion of RAFTINs. Scanning electron microscopy showed that
smaller in length (Fig. 9 A and B, which is published as
supporting information on the PNAS web site) and that the
pollen grains had collapsed (Fig. 9 C and D). There was no gross
difference in the pollen surfaces (Fig. 9 E and F). In pollen
germination assays, the average germination frequency of pollen
in the sterile lines was 4.7%, whereas that of the control lines was
74.3%. Thus, in the sterile lines, vegetative growth and flower
development in general were normal before anthesis, but pollen
grain development was apparently impaired.
Transmission electron microscopy showed no appreciable
differences between the sterile and control lines in terms of the
orbicular wall and Ubisch bodies of the sporophyte or the exine
of the gametophyte. Furthermore, in all these lines, microspore
development was typical (28) up to the free young microspore
stage. In the control line, the tapetum showed signs of degen-
eration at the vacuolated microspore stage and the microspore
continued its rapid expansion. During the subsequent stages, the
tapetum degenerated to release its metabolites for microspore
development and the microspore underwent sequential mitotic
divisions and developed into tri-nucleate pollen grains rich in
starch granules and other cytoplasmic contents (Fig. 3 A and B).
The tapetal degeneration proceeded to completion, showing few
remnants. In contrast, the vacuolated microspores of the osRAF-
TIN1-silenced lines started to collapse and the tapetum did not
initiate degeneration at the end of the vacuolated microspore
haipin RNA strategy. Northern blot analysis of osRAFTIN1 expression in trans-
genic rice transformed with intron-containing hairpin constructs. Percentage
(control) normalized against total RNA loaded is given. The numbers above
the lanes refer to the pAMW constructs listed in Fig. 7.
Silencing osRAFTIN1 in transgenic rice by using an intron-containing
osRAFTIN1-silenced rice (line 507-5) and control (empty vector alone). (A)
Mature anther from the control line. (B) Enlargement of A. (C–F) Anther from
line 507-5 at different developmental stages. (C) Free microspore stage. (D)
Vacuolated microspore stage. (E) Mature stage. (F) Enlargement of E. The
arrows in D point to the microspores that show signs of collapsing and those
in E and F point to the appressed exines of collapsed microspores. en, endo-
thecium; ep, epidermis; ex, exine; ms, microspore; ob, orbicular wall; ta,
tapetum; ub, Ubisch body. (Scale bars ? 10 ?m.)
Wang et al.
November 25, 2003 ?
vol. 100 ?
no. 24 ?
stage (Fig. 3D). The collapsed microspores appeared as flat
pollen grains (Fig. 3E). The tapetal degeneration was arrested
apparently at the vacuolated microspore stage, leaving the partly
the endothecium wall had thickened (Fig. 3 E and F).
RAFTIN1 Gene Expression and Protein Localization Suggest a Trajec-
tory from the Tapetum to Microspores via the Ubisch Bodies. The
anther comprises differentiated tissues such as the epidermis,
endothecium, middle layer, tapetum, and other supportive tis-
sues, and developing microspores. In both wheat and rice, in situ
hybridization localized RAFTIN1 mRNA distinctly to the tape-
tum and not to the filament, anther wall, or microspores (Fig. 4).
Extended hybridization caused high background reaction with-
out enhancing any specific signal in the microspores, suggesting
that if there was any expression in the microspores it was not
kDa) only in young florescence or anther but not in root, stem,
leaf, or emasculated florescence (Fig. 5A). In situ immuno-
microspore as well (Fig. 5B). RAFTIN was not detectable in
other tissues. The presence of RAFTIN was evident from the
early free microspore stage (Fig. 5E), becoming abundant in
both the tapetum and the microspores as the latter underwent
rapid expansion (Fig. 5 E–G). The RAFTIN signal decreased in
the three-nucleate pollen grain stage (Fig. 5H). These results
suggested temporal and spatial control of RAFTIN production
and deposition. Immunogold-labeled antibodies localized
(Fig. 6; controls are shown in Fig. 10, which is published as
supporting information on the PNAS web site). In the micro-
spores, RAFTINs were found to be dispersed in the tectum,
baculum, and foot layer (Fig. 6C). We examined overlapping
field views of the microspore cytosol in independent sections but
did not find a similar occurrence of labeled RAFTINs; a rare
section that did have a few gold particles is included in Fig. 6E.
This indicates very little expression, if any, of RAFTIN genes in
the microspore. For a given surface area in the electronmicro-
graphs, the ratio of gold particles was 44 for the exine, 29 for the
Ubisch bodies, and 17 for the tapetum in comparison with the
microspore cytosol as the reference at 1 (0.09 particles per ?m2
of microspore cytosol area). It has been suggested that Ubisch
bodies transport sporopollenin from the tapetum to the devel-
oping microspores of grasses and thus are involved in the
formation of the sporoderm (15). Accordingly, the locales of
RAFTIN suggest that RAFTIN is synthesized in the tapetum,
packaged in Ubisch bodies, and transported at appropriate
developmental stages to the microspores. Because the Ubisch
bodies also contain electron-opaque sporopollenin, it will be
interesting to see how RAFTIN is trafficked. Further studies
would be required to elucidate the pathways of RAFTIN
Organization of RAFTIN1 Genes and Gene Products. The features of
the genes are depicted in Fig. 11, which is published as support-
ing information on the PNAS web site. taRAFTIN1a and
taRAFTIN1b ORFs were predicted to encode a 389-aa and a
362-aa product, respectively. These two ORFs were 89% iden-
tical to each other at the nucleotide level and 86% at the amino
acid level and were derived from three exons. taRAFTIN1a
includes a near-perfect tandem repeat of 21 aa from the 96th
amino acid, but the corresponding region of taRAFTIN1b lacks
much of the amino acid in these repeats (Fig. 12A, which is
published as supporting information on the PNAS web site).
Additional cDNA clones that were distinct from the above were
not identified in three further screens of the anther cDNA
library, even though a shorter genomic fragment (named
taRAFTIN1c) from hexaploid wheat that was ?96% identical
to taRAFTIN1a and taRAFTIN1b had been isolated by PCR.
taRAFTIN1c was not pursued further. The predicted ORF of
osRAFTIN1, also composed of three exons, encodes a 412-aa
polypeptide that shares ?62% identity with its wheat counter-
parts over the entire length. Southern hybridization of genomic
DNA from rice (a diploid) and wheats of hexaploid (AABBDD),
to a taRAFTIN1a sense probe (control). (Scale bars ? 100 ?m.)
In situ RNA hybridization of RAFTIN1 transcripts in cross sections of
antibodies (B) and preimmune sera (control) (C). (D–H) Immunocytochemical detection of RAFTIN protein at different anther development stages. (D) ‘‘Tetrad’’
stage. (E) ‘‘Free microspore’’ stage. (F) ‘‘Vacuolated microspore’’ stage. (G) ‘‘Vacuolated pollen grain’’ stage. (H) ‘‘Three-nucleate pollen grain’’ stage. Root, root
tissue; Stem, stem tissue; Leaf, leaf tissue; Flower, developing young flower tissue; Fl w?o anther, developing young flower tissue with anther removed; Anther,
bar ? 40 ?m.)
Detection of RAFTIN proteins in wheat anther. (A) Western blot analysis of RAFTIN1 in wheat tissues. The arrow points to a polypeptide of ?40 kDa,
www.pnas.org?cgi?doi?10.1073?pnas.2231254100 Wang et al.
tetraploid (AABB), or diploid (AA or DD) genome suggests the
presence of only one RAFTIN1 gene per haploid genome
equivalent (Fig. 11B).
The wheat and rice RAFTINs contain two predicted trans-
membrane domains (Fig. 12A). TBLASTNanalysis did not identify
any sequences similar to the 168-aa amino domain of
taRAFTIN1a, with the exception of ESTs from cereals and a
chromosome segment of rice. The remainder (carboxyl domain
of 221 aa) was 77% identical to the corresponding portion of
osRAFTIN1 (Fig. 12A) and ?90% identical to ESTs from
inflorescences of various cereal species. All other significant
‘‘hits’’ (E value of ?10?4) were also only from plant accessions,
but generally they had only ?35% identity and included the
following BURP domain proteins (29): a seed protein of faba
bean (30); the ? subunit of polygalacturonase isoenzyme 1 from
the developing fruit of tomato (31); an desiccation-induced
protein of Arabidopsis (32); an auxin down-regulated protein
(33) and an aluminum up-regulated protein (34) from soybean
hypocotyl and roots, respectively; microspore embryo develop-
ment-related protein from Brassica napus (29); an apomixis-
specific gene product from the flower buds of guinea grass (35)
that showed ?45% aa sequence identity over the entire carboxy-
domain; and a seed coat protein found in developing soybean
seed coats (36).
We have identified a tapetal protein that packages into Ubisch
bodies and microspore exine, and this protein, RAFTIN, from
wheat and rice has counterparts only in cereal species. RAFT-
INs, however, have the features of the enigmatic, plant-specific
group of proteins that have been named as BURP domain
proteins (29). The most striking commonality of the latter is the
presence of ?30 conserved residues that are dispersed over the
carboxyl region of ?220 aa; the amino region is not conserved
and its length is highly variable (68–414 aa). Two C and four CH
residues among the 30 conserved amino acids are the hallmarks
of the so-called BURP signature. The amino termini in all these
proteins also include a predicted signal peptide region. Deduced
BURP domains have been noted in genomic?cDNA?EST se-
quences from both monocots and dicots, and the expression
patterns of the corresponding genes include vastly distinct
developmental contexts. The essentiality of these proteins to
plant form or function has not been previously demonstrated.
maturation phase of pollen development.
Pollen development may be generalized to occur through the
following stages: meiosis?tetrad, young free microspore, vacu-
olated and expanded microspore, vacuolated pollen grain and
three-nucleate pollen grain. The tapetum provides nutrients,
proteinaceous and lipidic precursors for developing microspores
and it secretes the callase that is required for resolving the
postmeiotic tetrads (12, 18, 20, 37–39). Continuing with the
supportive role, the tapetum ultimately degenerates in a pro-
grammed manner at the advanced stages of microspore devel-
opment, releasing its contents into the locule and leaving behind
the remnants and orbicular wall-attached Ubisch bodies (28). It
fertility and afford specificity in pollen–pistil interactions (40–
43). In Brassicaceae members, proteins and lipids from tapetum
become a part of the pollen exine and the outer coatings (20, 44).
There are no Ubisch bodies in Brassicaceae (15), and some of the
tapetal lipids and proteins are packaged into tapetosomes and
elaioplasts (21, 45). These organelles have not been described in
wheat or rice, and the transport of tapetal contents by mode(s)
other than secretion or tapetal lysis has not been shown for these
tapetum and they are extruded to locular side where the cores
become surrounded by sporopollenin polymer, which is similar
Magnified views of the boxed zones from A. (F) Diagrammatic representation of an anther cross section pointing out the zones in B–E and a key for the
abbreviations. Arrows point to gold particles. (Scale bar ? 3 ?m in A and 0.5 ?m in B–E.)
Wang et al.
November 25, 2003 ?
vol. 100 ?
no. 24 ?
to the exine- and peritapetal wall-associated sporopollenin in
electron-opacity (12, 17, 46, 47). Ubisch bodies have not been
isolated from any species. Although the Ubisch body-associated
RAFTIN is surmised to be in transit from the tapetal cells to the
microspore exine, independent embedding of RAFTINs within
the sporopollenin matrices of Ubisch bodies and the exine
cannot be ruled out.
Sporopollenin comprises phenylpropanoids and fatty acid
derivatives, but its exact composition is unknown (48). It is
synthesized in young microspores and also in the sporophytic
tapetum. The microspore-synthesized sporopollenin is used for
exine formation in the early stages when the microspores are
shielded from the locular fluid by a callose wall, and exine
maturation occurs in conjunction with deposition of tapetally
derived sporopollenin (17, 48). The physico-biochemical aspects
of the transformation of sporopollenin as a recalictrant polymer
remain to be elucidated. We have found RAFTINs in orbicular?
peritapetal wall, Ubisch bodies and the exine, the three locations
where polymerized sporopollenin occurs. Based on the spatio-
wall and the exine with reference to pollen development (17, 47),
and the collapse of late-stage microspores in RAFTIN-silenced
lines, it seems possible that RAFTINs could play a guiding role
in proper ‘‘fixation’’ of sporopollenin polymers from its precur-
sors of tapetal origin. The lack of any gross changes in the
electron-opacity of the exine of the collapsed microspores
suggests that the changes in the exine are subtle and yet the
effects on microspore maturation beyond the stages of cell
vacuolation and expansion are dramatic. A gametocidal agent
that affects sporopollenin deposition has a similar effect on
wheat microspores (12). Clearly, the formation of nearly normal
vacuolated microspores in the RAFTIN-silenced lines shows that
the gametophytic contribution to exine ontogeny is unaffected
and that RAFTIN has a role pertaining to maturation.
We gratefully acknowledge the assistance of Sara R. Caldwell and
Elfriede Moya with microscopy; Rozina Hirji and Eugen Kurylo with
EST generation; Kevin Koh with EST analysis; Ping Zheng with rice
transformation; Darrin Klassen, Barry Panchuk, and Inge Roewer with
DNA sequencing; and Don Schwab with oligonucleotide synthesis. We
thank Dayakar Pareddy and Tonya Strange for advice on rice transfor-
mation, Dr. Takuji Sasaki for providing the initial rice seed stock, and
the anonymous reviewers for their very helpful comments. This work was
funded by the Strategic Initiatives Fund and the National Research
Council of Canada. This is National Research Council of Canada
1. Koltunow, A. M., Truettner, J., Cox, K. H., Wallroth, M. & Goldberg, R. B.
(1990) Plant Cell 2, 1201–1224.
2. Bedinger, P. A. (1992) Plant Cell 4, 879–887.
3. Chaudhury, A. M. (1993) Plant Cell 5, 1277–1283.
4. Goldberg, R. B., Beals, T. P. & Sanders, P. M. (1993) Plant Cell 5, 1217–1229.
5. Hamilton, D. A. & Mascarenhas, J. P. (1997) in Pollen Biotechnology for Crop
Production and Improvement, eds. Shivanna, K. R. & Sawhney, V. K. (Cam-
bridge Univ. Press, Cambridge, U.K.), pp. 41–58.
6. McCormick, S. (1993) Plant Cell 5, 1266–1275.
7. Shivanna, K. R., Cresti, M. & Ciampolini, F. (1997) in Pollen Biotechnology for
Crop Production and Improvement, eds. Shivanna, K. R. & Sawhney, V. K.
(Cambridge Univ. Press, Cambridge, U.K.), pp. 15–39.
8. Yang, W. C. & Sundaresan, V. (2000) Curr. Opin. Plant Biol. 3, 53–57.
9. D’Arcy, W. G. (1996) in The Anther: Form, Function, and Phylogeny, eds.
D’Arcy, W. D. & Keating, R. C. (Cambridge Univ. Press, Cambridge, U.K.),
10. De Block, M., Debrouwer, D. & Moens, T. (1997) Theor. Appl. Genet. 95,
11. Mariani, C., Gossele, V., de Beuckeleer, M., de Block, M., Goldberg, R. B., de
Greef, W. & Leemans, J. (1992) Nature 357, 384–387.
12. Mizelle, M. B., Sethi, R., Ashton, M. E. & Jensen, W. A. (1989) Sex. Plant
Reprod. 2, 231–253.
13. Bhojwani, S. S. & Bhatnagar, S. P. (1992) The Embryology of Angiosperms
(Vikas, New Delhi), pp. 11–32.
14. Furness, C. A. & Rudall, P. J. (2001) Int. J. Plant Sci. 162, 375–392.
15. Huysmans, S., El-Ghazaly, G. & Smets, E. (1998) Bot. Rev. 64, 240–272.
16. Heslop-Harrison, J. (1968) Science 161, 230–237.
17. Dickinson, H. G. & Bell, P. R. (1972) Planta 107, 205–215.
18. Sanders, P. M., Bui, A. Q., Weterings, K., McIntire, K. N., Hsu, Y.-C., Lee,
P. Y., Truong, M. T., Beals, T. P. & Goldberg, R. B. (1999) Sex. Plant Reprod.
19. Piffanelli, P., Ross, J. H. E. & Murphy, D. J. (1997) Plant J. 11, 549–652.
21. Wu, S. S. H., Platt, K. A., Ratnayake, C., Wang, T.-W., Ting, J. T. L. & Huang,
A. H. (1997) Proc. Natl. Acad. Sci. USA 94, 12711–12716.
22. Wang, A., Xia, Q., Xie, W., Dumonceaux, T., Zou, J., Datla, R. & Selvaraj, G.
(2002) Plant J. 30, 613–623.
23. Chen, L., Zhang, S., Beachy, R. N. & Fauquet, C. M. (1998) Plant Cell Rep. 18,
24. Wang, A. & Sanfacon, H. (2000) J. Gen. Virol. 81, 2771–2781.
25. Wan, L., Xia, Q., Qiu, X. & Selvaraj, G. (2002) Plant J. 30, 1–10.
W., Martienssen, R., Selvaraj, G. & Datla, R. (2002) Proc. Natl. Acad. Sci. USA
27. Smith, N. A., Singh, S. P., Wang, M.-B., Stoutjesdijk, P. A., Green, A. G. &
Waterhouse, P. M. (2000) Nature 407, 319–320.
28. Raghavan, V. (1988) Am. J. Bot. 75, 183–196.
29. Hattori, J., Boutilier, K. A., van Lookeren Campagne, M. M. & Miki, B. L.
(1998) Mol. Gen. Genet. 259, 424–428.
30. Ba ¨umlein, H., Boerjan, W., Nagy, I., Bassu ¨ner, R., Van Montagu, M., Inze ´, D.
& Wobus, U. (1991) Mol. Gen. Genet. 225, 459–467.
31. Zheng, L., Heupel, R. C. & DellaPenna, D. (1992) Plant Cell 4, 1147–1156.
32. Yamaguchi-Shinozaki, K. & Shinozaki, K. (1993) Mol. Gen. Genet. 238, 17–25.
33. Datta, N., LaFayette, P. R., Kroner, P. A., Nagao, R. T. & Key, J. L. (1993)
Plant Mol. Biol. 21, 859–869.
34. Ragland, M. & Soliman, K. M. (1997) Plant Physiol. 114, 395.
35. Chen, L., Miyazaki, C., Kojima, A., Saito, A. & Adachi, T. (1999) J. Plant
Physiol. 154, 55–62.
36. Batchelor, A. K., Boutilier, K., Miller, S. S., Hattori, J., Bowman, L. A., Hu, M.,
Lantin, S., Johnson, D. A. & Miki, B. L. A. (2002) Planta 215, 523–532.
37. Cle ´ment, C., Laporte, P. & Audran, J. C. (1998) Sex. Plant Reprod. 11, 94–106.
39. Pacini E. (1990) in Microspores: Evolution and Ontogeny, eds. Blackmore, S. &
Knox, R. B. (Academic, London), pp. 213–237.
40. Mayfield, J. A., Fiebig, A., Johnstone, S. E. & Preuss, D. (2001) Science 292,
41. Pruitt, R. E., Vielle-Calzada, J.-P., Ploense, S. E., Grossniklaus, U. & Lolle,
S. J. (2000) Proc. Natl. Acad. Sci. USA 97, 1311–1316.
42. Stintzi, A. & Browse, J. (2000) Proc. Natl. Acad. Sci. USA 97, 10625–10630.
43. Wolters-Arts, M., Lush, W. M. & Mariani, C. (1998) Nature 392, 818–821.
44. Staigher, D., Kappeler, S., Mu ¨ller, M., Apel, K. (1994) Planta 192, 221–231.
45. Herna ´ndez-Pinzo ´n, I., Ross, J. H. E., Barnes, K. A., Damant, A. P. & Murphy,
D. J. (1999) Planta 208, 588–598.
46. El-Ghazaly, G. & Jensen, W. A. (1986) Grana 25, 1–29.
47. Heslop-Harrison, J. & Dickinson, H. G. (1969) Planta 84, 199–214.
48. Scott, R. J. (1994) in Molecular and Cellular Aspects of Plant Reproduction, eds.
Scott, R. J. & Stead, A. D. (Cambridge Univ. Press, Cambridge, U.K.), pp.
www.pnas.org?cgi?doi?10.1073?pnas.2231254100Wang et al.