Molecular cloning and characterization of
OsLRK
1 encoding a
putative receptor-like protein kinase from
Oryza sati
6
a
Chanhong Kim, Dong-Hoon Jeong, Gynheung An *
Department of Life Science,Pohang Uni6ersity of Science and Technology,Pohang790-784,South Korea
Received 13 September 1999; received in revised form 15 September 1999; accepted 16 September 1999
Abstract
A cDNA clone, OsLRK1, encoding a leucine-rich repeat receptor-like protein kinase was isolated from the immature panicles of rice. The OsLRK1 protein was composed of a leucine-rich extracellular ligand-binding domain, a membrane-spanning segment, and a cytoplasmic kinase domain. The OsLRK1 protein showed the highest sequence homology with Arabidopsis CLV1. The
OsLRK1 transcript was present at a high level in immature panicles and at a low level in seedling shoots, immature seeds, and mature seeds, while no expression was detected in seedling roots and panicles at the heading stage. This expression pattern is similar to that ofCLV1, which suggests that the rice clone may play a critical role in meristem development. The function of the riceOsLRK1 gene was studied by the transgenic approach. Antisense expression of OsLRK1 in rice plants resulted in increased numbers of flower organs, suggesting that the OsLRK1 gene is involved in floral meristem activity. © 2000 Elsevier Science Ireland Ltd. All rights reserved.
Keywords:Antisense; CLAVATA1; Floral meristem; Leucine-rich repeats; Receptor kinase; Rice transformation
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1. Introduction
Plant development requires the integration of various signaling pathways that recognize and re-spond to endogenous and exogenous information. Plants perceive and respond to various stimuli such as hormones, pathogens, nutrients, light, and stress [1 – 5]. However, the mechanisms by which plants detect and transduce these signals into their cells are poorly understood. Recent evidence sug-gests that plants have many different types of transmembrane protein kinases that may function to transduce extracellular information into the cell [6 – 8]. Some of these plant proteins are called receptor-like protein kinases (RLKs).
RLKs appear to serve as receptors for extracel-lular signals that are involved in processes such as plant growth, development, and defense. The binding of a signal ligand to an RLK may result in
dimerization and activation, leading to a cellular response [9]. RLKs have been identified from a number of plants and have been categorized into classes based on various structural motifs found in their extracellular domains [10]. They contain a cytoplasmic protein kinase domain that is acti-vated when a ligand binds to the extracellular receptor domain.
Recent studies have placed RLKs within various signaling cascades, including those for pathogen response, developmental processes, and hormone response [11]. TheArabidopsis CLAVATA(CLV)1 gene, which encodes such a receptor kinase, is required for normal development of the shoot apical and floral meristem [1]. In cl61 mutants, extended floral meristem activity results in extra carpel primordia being initiated inside the gynoe-cium [12]. The enlarged meristems, which have abnormally large pools of undifferentiated and proliferative cells, give rise to an increased number of flowers (from the inflorescence meristem) and floral organs (from the floral meristem) [1,12].
* Corresponding author. Tel.: +82-562-279-3176; fax: + 82-562-279-2199.
E-mail address:[email protected] (G. An)
The characterization of the process from floral meristem formation to floral organ primordia for-mation is an important part of the analysis of flower development. However, very little is known about the mechanisms controlling how cell fates are determined in the floral meristem. Cell fate selections include whether to form an organ pri-mordium, which type of organ to differentiate into, and when to terminate cell proliferative and organogenic activities. Because cell fate selection in the inflorescence and floral meristems is likely to be a very complex process, it is also likely that many genes are involved in this process. Analysis of novel floral regulatory genes will provide new insights into flower development. Much of the studies have been focused on dicot species. In order to understand flower development in cereal plant species, we have cloned and characterized a receptor kinase gene, OsLRK1, from rice
2. Materials and methods
2.1. Bacterial strains
Escherichia coliJM 83 was used as the recipient for routine cloning experiments. Agrobacterium tumefaciens LBA4404 [13] containing the Ach5 chromosomal background and a disarmed helper-Ti plasmid pAL4404 was used for transformation of rice. The f1 helper phage R408 and E. coli strain XL-1Blue were used for in vivo excision of the pBluescript plasmid vector from the lZAPII phage (Stratagene, CA).
2.2. Screening of cDNA library and sequence analysis
A partial clone of the EST (expressed sequence tag) C22553 (GenBank accession number) was isolated by PCR (polymerase chain reaction) us-ing the primers, 5%
-GAGCTTCTCCGGT-GTTCAG-3% and
5%-GATGTGGCATTGAAGTAGCT-3%. A cDNA library was constructed from mRNA prepared from young panicles (length under 2 cm). Hy-bridization was performed with 320 000 plaques using the [a-32P] dCTP labeled probe of the par-tial cDNA clone of OsLRK1 by the random priming method [14]. The nucleotide sequence was determined using an automated sequencer (ABI 373A). DNA sequence comparison was
per-formed using the DDBJ (DNA data bank of Japan) database.
2.3. RNA isolation and RNA gel blot analysis
Total RNA was extracted from various tissues of rice plants using the RNA isolation kit (Tri reagent, Molecular Research Center, INC). Elec-trophoresis of total RNA (10 mg) was carried out in a 1.2% (v/v) formaldehyde – agarose gel as de-scribed previously [14]. The RNA was blotted onto a nylon membrane (Hybond-N+, Amer-sham) and hybridized in a solution containing 0.5 M sodium phosphate (pH 7.2), 1 mM EDTA, 1% BSA, and 7% SDS for 20 h at 55°C [15]. After hybridization, the blot was washed twice with a solution containing 0.1×SSPE and 0.1% SDS for 5 min at room temperature, followed by two washes of the same solution at 55°C for 20 min. Hybridization was performed with the [a-32P] dCTP labeled probe of the partial cDNA clone of OsLRK1 by the random priming method [14].
2.4. Construction of binary 6ectors
A binary vector, pGA1611, that can be used for transformation of rice plants was constructed. This vector, a derivative of pGA482 [16], contains the hygromycin phosphotransferase (hph) gene as a selectable marker under the control of the cauliflower mosaic virus 35S promoter followed by the termination region of the 7 gene of pTiA6. The vector also contains several unique sites (HindIII, SacI, HpaI, and KpnI) between the maize ubiquitin promoter, including the first in-tron of the ubiquitin gene [17], and the nopaline synthase (nos) terminator. Therefore, this vector can be used for expression of a foreign gene into monocot plants when transferred by the Agrobac-terium co-cultivation method. The partial cDNA clone of OsLRK1 was inserted into the multiple cloning sites in an antisense orientation, con-structing pGA2152.
2.5. Rice transformation
medium containing 2 mg/l 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 mg/l hygromycin and 3 mg/l tetra-cycline. Three-week-old calli were co-cultivated with the Agrobacterium on a 2N6-As medium supplemented with 100mM betaine for 2 – 3 days in darkness at 25°C. The co-cultivated calli were washed with sterile water containing 100 mg/l cefotaxime and incubated on an N6 medium con-taining 40 mg/l hygromycin and 250 mg/l cefo-taxime for 3 weeks. Actively growing calli were transferred onto a regeneration medium, MS medium supplemented with 0.1 mg/l NAA, 2 mg/l kinetin, 2% sorbitol, 1.6% phytagar (Sigma), 50 mg/l hygromcyin, and 250 mg/l cefotaxime. After 2 – 3 weeks under continuous light (40mmol/m 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 OsLRK1 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 toCLV1 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-S/T). 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 serine/threonine 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 OsLRK1
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 OsLRK1 construct
In order to elucidate roles of the OsLRK1 gene, the 387-bp DNA fragment of the partial
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
in reproductive organs, including palea/lemmas, lodicules, stamens, and carpel.
3.4. RNA gel blot analysis of the OsLRK1 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 (c4 and c5) 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
Fig. 4. Expression pattern of theOsLRK1 transcript in differ-ent organs (A) and developing panicles (B). (A) Ten 10mg 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 (B2 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 paleas/lemmas (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 palea/lemma 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 palea/lemma and lodicule.
Fig. 5. Expression pattern of the OsLRK1 transcript in the transgenic plants. Panicles (B5 cm) from wild type (A) or transgenic plant line c4 (B) and c5 (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 OsLRK1 transgenic plants
Fig. 6. Phenotypes of the transgenic flowers expressing the antisenseOsLRK1 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 linec4 flower with one extra palea/lemma. (C) Transgenic linec5 flower with two extended lodicules. (D) Transgenic line c4 flower with two extra lodicules. (F) Transgenic line c5 flower with two extra stamens and one extra carpel. G, transgenic line c4 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 andOsLRK1 transgenic plants
Palea or lemmab
Plant Frequencya Lodiculesb Stamensb Carpelsb
2.090 2.090
0/23 6.090
Wild type 1.090
Transgenic line c4 17/25 3.190.4 2.990.7 7.090.7 1.590.5 3.190.2 3.290.5 6.990.6
17/23 1.490.4
Transgenic line c5
aNumber of abnormal flowers/number of flower examined. bIn transgenic lines, number of floral organs in abnormal flowers.
4. Discussion
Flowers develop from groups of undifferenti-ated cells on the flanks of the reproductive shootapical meristem. Initially, these cells form the floral meristem. The cells on the floral meristem then develop with different fates to generate the appropriate numbers of floral organs in the appro-priate place [23].
second whorl, stamens in the third whorl, and carpels in the fourth whorl. Therefore, cells in certain regions of the floral meristem must cor-rectly interpret their positional information and then organize into specific types of organ primordia.
Molecular and genetic studies of flower develop-ment have led to the identification and characteri-zation of a number of genes that affect the pathway from inflorescence meristem formation to floral organ primordium formation [25 – 27]. Among these genes, LEAFY (LFY) and APETALA1 (AP1) are involved in the formation of floral meristem from the inflorescence meristem [28,29]. Both thelfy andap1 mutants show partial transformations of flowers into inflorescence shoots.
Another gene that affects the floral meristem as well as the apical meristem is CLV1. It has been reported thatcl61 mutants exhibit a variable num-ber of extra organs in each whorl [30]. TheCLV1 gene is expressed in aboveground meristems, and it has been proposed that role of the gene is to restrict both shoot meristem and floral meristem activities [1]. cl61 mutants initiate flowers with an increased organ number, especially in the inner whorl of carpels, and they have larger shoots and floral meristems. The increase in flower organ number could be due either to CLV1 negatively regulating the proliferation of central zone cells or to CLV1 promoting organogenesis in the periph-eral zone.
In this study, we have isolated the OsLRK1 cDNA from immature panicles of rice. The pre-sumed extracellular domain is composed almost exclusively of LRRs, which are known to be in-volved in protein – protein interactions [6]. At least half of the known LRR-containing proteins par-ticipate in signal transduction [8]. When the amino acid sequences of the LRR domain and the kinase domain of OsLRK1 were compared separately on the DDBJ database, out of the RLKs with known function, they were most homologous with CLV1. In addition, both CLV1 and OsLRK1 contained a similar conserved LRR domain structure, with 21 tandem LRR repeats. On the amino acid level, OsLRK1 shares the highest identity to CLV1, and within the conserved LRR domain, it was also most similar to CLV1 (Fig. 2). The OsLRK1 gene was preferentially expressed in immature (B2 cm) and young panicles (2 – 5 cm). The gene was also
expressed in seedling shoots containing a shoot meristem, but at a lower level, and it is not detected in roots at all. This expression pattern of OsLRK1 highly resembles that of CLV1.
We have studied the roles of the rice OsLRK1 gene by the loss-of-function approach.
In the experiments using antisense suppression of the OsLRK1 gene, expression of the OsLRK1 transcript was completely absent in some trans-genic plants, whereas the level of other cross-hy-bridized transcripts was not affected in the plants. The 387-bp DNA fragment that was used for generation of antisense transgenic plants contained a portion of the LRR domain, but the more conserved kinase domain was not included to in-crease specificity. It is generally accepted that a high degree of sequence homology is needed for antisense suppression. Therefore, it is likely that the antisense construct preferentially inhibited ex-pression of OsLRK1.
The phenotypes displayed by the antisense OsLRK1 transgenic plants described here proba-bly resulted from meristem enlargement and shape change. This in turn altered the pattern of organ initiation. The most phenotypic alteration was ob-served in the outer two whorls, the palea/lemma and the lodicules, although OsLRK1 affects the whole floral meristem. This phenotype is different from that of cl61 mutants, which displayed alter-ation of floral organ number mainly in carpel [30]. In addition the cl61 mutants showed increase in number of flowers. However, the number of flow-ers in the OsLRK1 transgenic rice plants was not affected, suggesting that OsLRK1 was active mainly in the floral meristem. These suggest that OsLRK1 is a member of the CLV family, but OsLRK1 is functionally distinct from CLV1.
Acknowledgements
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![Fig. 3. Multiple alignment of kinase domain. Alignment of the kinase domains among several putative LRR receptor kinases inplants including CLV1, ERECTA, BRI, and Xa21 [1–4]](https://thumb-ap.123doks.com/thumbv2/123dok/1035101.928979/6.612.103.456.258.646/multiple-alignment-alignment-putative-receptor-inplants-including-erecta.webp)
