medium containing 2 mgl 2,4-D. A. tumefaciens strain LBA4404 carrying the pGA2152 plasmid was
grown for 3 days in an AB liquid medium supple- mented with 15 mgl hygromycin and 3 mgl tetra-
cycline. Three-week-old calli were co-cultivated with the Agrobacterium on a 2N6-As medium
supplemented with 100 mM betaine for 2 – 3 days in darkness at 25°C. The co-cultivated calli were
washed with sterile water containing 100 mgl cefotaxime and incubated on an N6 medium con-
taining 40 mgl hygromycin and 250 mgl cefo- taxime for 3 weeks. Actively growing calli were
transferred onto a regeneration medium, MS medium supplemented with 0.1 mgl NAA, 2 mgl
kinetin, 2 sorbitol, 1.6 phytagar Sigma, 50 mgl hygromcyin, and 250 mgl cefotaxime. After
2 – 3 weeks under continuous light 40 mmolm per s, the plantlets were transferred to soil and grown
in a growth chamber with 10 h light per day.
3. Results
3
.
1
. Isolation of OsLRK
1
encoding a putati6e leucine-rich repeat receptor kinase from rice
In order to isolate a CLV1 homolog from rice, we searched the rice DDBJ database using the
CLV1 extracellular domain sequence as a query. Several ESTs showed homology to CLV1 and these
clones contained the conserved residues found in the leucine-rich repeat LRR domain of CLV1.
Among these ESTs, C22553 showed the highest identity 49. The 387-bp partial clone of the EST
carrying a portion of the LRR domain was isolated by PCR using gene specific primers and the first
strand cDNA of immature rice panicles as a tem- plate.
A full-length cDNA clone was isolated from the immature panicle cDNA library with the 387-bp
partial clone as a probe. Screening 320 000 plaques resulted in 77 positive signals. Among these clones,
nine were rescued in vivo and further characterized. Restriction enzyme mapping of the inserts and
partial sequencing of the ends revealed that these clones could be grouped into five independent
cDNAs. One of the clones that showed significant homology to Arabidopsis CLV1 and contained the
entire coding region was selected for further study. This clone was named OsLRK1 Oryza sati6a
leucine-rich repeat receptor-like kinase 1. The cDNA clone is 3.5-kb long, containing an
open reading frame of 971 amino acid residues with a 93-bp 5 untranslated region and a 539-bp 3
non-coding region Fig. 1. The amino terminus of the OsLRK1 polypeptide has a putative signal
peptide sequence that may direct secretion [20]. This is followed by a potential extracellular domain
consisting of a conserved LRR region between the amino acid residues 89 and 599. The LRR domain
region consists of 21 tandem copies of 24-amino acid LRRs Fig. 2 with N-linked glycosylation
consensus sites N-X-ST. This region shares 49, 34, 37, and 34 amino acid sequence identities with
the LRR domain in CLV1, ERECTA, BRI1, and Xa21, respectively. The LRR region is flanked by
pairs of conservatively spaced cysteines. The LRR domain is followed by a stop-transfer sequence that
is rich in charged amino acids, suggesting that it is a transmembrane domain amino acids 647 – 666
[21]. The putative intracellular domain contains all of the 12 subdomains and the conserved residues
found among serinethreonine protein kinases Fig. 3 [22].
Based on amino acid sequence similarity, it can be concluded that OsLRK1 is a member of the
LRR receptor kinase family. Among the LRR receptor kinases with a known biological function,
the OsLRK1 protein revealed the highest sequence identity to Arabidopsis CLV1. The OsLRK1 and
CLV1 show 55 amino acid sequence identity over their entire coding regions and 72 in the kinase
domains.
3
.
2
. Expression pattern of OsLRK
1
Total RNAs were extracted from various organs at different developmental stages, and the tran-
script levels of the OsLRK1 genes were determined by RNA blot analysis using a probe prepared from
the 387-bp partial clone carrying a portion of the LRR domain. The 3.5-kb OsLRK1 transcript was
detected at a high level in young panicles. Lower level of the transcript was detected in shoots of
7-day-old seedling, immature seeds 2 – 5 days after pollination, and young seeds 8 – 10 days after
pollination. However, the transcript was not de- tectable in seedling roots and panicles at the head-
ing stage Fig. 4A. Expression of the OsLRK1 gene was also studied during panicle development. The
OsLRK1 transcript level was high in immature
panicles at early developmental stages, and the transcript level decreased as the panicles devel-
oped Fig. 4B. These data suggestthat OsLRK1 may be preferentially expressed in the above-
ground meristem tissues. It is likely that the weakly hybridizing band smaller than the 3.5-kb
transcript is a product of an OsLRK1-related gene since the level of the smaller band was not
affected by antisense suppression of OsLRK1 see below.
3
.
3
. Transformation of rice plants with the antisense OsLRK
1
construct In order to elucidate roles of the OsLRK1
gene, the 387-bp DNA fragment of the partial
Fig. 1. Nucleotide and deduced amino acid sequences of the OsLRK1 cDNA. The regions corresponding to two conservatively spaced cysteine pairs are underlined. The potential N-linked glycosylation sites are in bold lettering and the predicted
transmembrane sequence is double underlined. The conserved kinase domains are boxed and indicated with Roman numerals. The shaded sequences are the primers used for generation of the 387-bp DNA fragment.
Fig. 2. Alignment of LRR repeats in the OsLRK1 protein. The shaded boxes indicate the residues that appear at each position at more than 50 frequency. The bottom is a comparison of the LRR consensus sequence of OsLRK1 with the consensus
sequences of other LRR-containing proteins.
OsLRK1 cDNA clone was placed in an antisense orientation under the control of the maize ubiquit-
inpromoter [17] and the nos terminator. The chimeric molecule, pGA2152, was introduced to
rice plants using the Agrobacterium-mediated transformation method. Forty independent hy-
gromycin-resistant transgenic plants were gener- ated. All of the transgenic plants showed normal
development at the vegetative stage. However, 17 transgenic plants exhibited abnormal phenotypes
in reproductive organs, including palealemmas, lodicules, stamens, and carpel.
3
.
4
. RNA gel blot analysis of the OsLRK
1
antisense transgenic plants Expression of the transgene was studied using
RNA samples prepared from leaves of the trans- genic plants showing the phenotypic changes.
Since the ubiquitin promoter was used for expres- sion of the transgene, the transcripts of the intro-
duced OsLRK1 would be detected in all plant organs.
All the
transgenic plants
expressed OsLRK1 although there was a variation in the
transcript level data not shown. Two transgenic lines c 4 and c 5 that showed the most severe
phenotypic changes contained the highest level of the antisense transcript in leaves. Expression of the
transgene and the endogenous OsLRK1 gene in the lines was also studied using RNA samples
prepared from developing panicles. Since a frag- ment of the OsLRK1 cDNA was used for the
antisense construct, the antisense transcript was much shorter than the entire full-length sense tran-
script. Result showed that the antisense transcripts were present in the transgenic plants but the tran-
scripts of endogenous OsLRK1 were appeared to be degraded Fig. 5. Transgenic plants that
showed a mild phenotype expressed a lower level of the antisense transcript data not shown. These
results implied that the phenotypic changes found in the transgenic plants were caused by the ab-
Fig. 3. Multiple alignment of kinase domain. Alignment of the kinase domains among several putative LRR receptor kinases in plants including CLV1, ERECTA, BRI, and Xa21 [1 – 4]. The 12 conserved protein kinase domains are indicated I to XI [22].
Residues that are conserved among at least three of the compared kinase subdomains are shaded. The 15 invariant amino acids present in all protein kinases are indicated by asterisks. Dashes designate gaps to allow maximum alignment.
Fig. 4. Expression pattern of the OsLRK1 transcript in differ- ent organs A and developing panicles B. A Ten 10 mg of
total RNA was isolated from different organs and hybridized with a probe generated from the 387-bp DNA fragment
carrying a portion of the LRR repeat region. From 1-week- old seedling plants, SS, shoots including the shoot meristem
and sheath; and SR, seedling roots. From 8-week-old plants, ML, mature leaves; IP, immature panicles smaller than 5 cm;
YP, panicles at 5 – 10 cm length stage; MP, mature panicles at heading stage; IS, immature seeds at 2 – 5 DAP; and YS,
young seeds at 8 – 10 DAP. B Sample 1, immature panicles B 2 cm including panicle primordia. Sample 2, immature
panicles 2 – 5 cm. Sample 3, mature panicles at heading stage. Equal amounts of total RNA loading were identified
with ethidium bromide staining of rRNAs.
two lodicules, and a pair of bract-like structures called the lemma and palea. In the transgenic
flowers, the number of each floral organ was in- creased: two – four paleaslemmas Fig. 6B, two –
four lodicules Fig. 6D, six – nine stamens Fig. 6F and G, and one – two carpels Fig. 6F and G.
Furthermore,
some flowers
had extended
lodicules, which were deformed into various ab- normal shapes that vaguely resembled palea
lemma-like structures Fig. 6D and G. However, these transgenic flowers did not exhibit any home-
otic changes.
The phenotypic alterations of the floral organs, shown by changes in floral organ number or
shape, differed from other flowers isolated from the same plant. The transgenic plants had both
normal and abnormal flowers Table 1. In addi- tion, each abnormal flower did not show the same
phenotypic alterations. In the floral organ number, the highest frequency of the change was seen in
the palealemma and lodicule. No flower changed exclusively in the stamen and carpel. In addition,
morphological alterations of the floral organ ap- peared only on the lodicules. These results suggest
that OsLRK1 is involved in controlling the num- ber of floral organs, in particular, the palealemma
and lodicule.
Fig. 5. Expression pattern of the OsLRK1 transcript in the transgenic plants. Panicles B 5 cm from wild type A or
transgenic plant line c 4 B and c 5 C were used for extraction of total RNA. The probe was identical to that used
in the Fig. 4.
sence or reduction of the endogenous OsLRK1 mRNA and that the degree of the phenotypic
alteration was proportional to the level of the antisense transcript.
3
.
5
. Phenotypic alteration of floral organs in OsLRK
1
transgenic plants The two transgenic plants, c 4 and c 5, that
displayed the most severe phenotypic alteration were selected for further studies. These lines
showed changes in the number of floral organs Fig. 6. The wild type rice flower Fig. 6A and E
consists of a carpel surrounded by six stamens,
Fig. 6. Phenotypes of the transgenic flowers expressing the antisense OsLRK1 compared with the wild type flower. Wild type rice flower A, E that is composed of a carpel c surrounded by six stamen s, two lodicule lo, and a pair of bract-like structures
called the lemma l and palea p. B Transgenic line c 4 flower with one extra palealemma. C Transgenic line c 5 flower with two extended lodicules. D Transgenic line c 4 flower with two extra lodicules. F Transgenic line c 5 flower with two extra
stamens and one extra carpel. G, transgenic line c 4 flower with three extended lodicules, three extra stamens, and one extra carpel. Palea and lemma were removed to visualize the inner organs D – G. Arrow heads indicate extra organs in transgenic
flowers.
Table 1 Number of flower organs in wild-type and OsLRK1 transgenic plants
Palea or lemma
b
Plant Lodicules
b
Frequency
a
Stamens
b
Carpels
b
2.0 9 0 2.0 9 0
023 6.0 9 0
Wild type 1.0 9 0
Transgenic line c 4 3.1 9 0.4
1725 2.9 9 0.7
7.0 9 0.7 1.5 9 0.5
3.1 9 0.2 3.2 9 0.5
6.9 9 0.6 1723
1.4 9 0.4 Transgenic line c 5
a
Number of abnormal flowers number of flower examined.
b
In transgenic lines, number of floral organs in abnormal flowers.
4. Discussion
