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Cloning and structural and expressional characterization of BcpLH gene preferentially expressed in folding leaf of Chinese cabbage * YU Xuhong (余旭红), PENG Jiesong (彭洁松), FENG Xianzhong (冯献忠), YANG Suxin (杨素欣), ZHENG Zhiren (郑志仁), TANG Xiangrong (唐向荣), SHEN Ruijuan (沈瑞娟), LIU Pinglin (刘平林) & HE Yuke (何玉科) State Key Laboratory of Plant Molecular Genetics, Shanghai Institute of Plant Physiology, Chinese Academy of Sciences, Shanghai 200032, China Correspondence should be addressed to He Yuke (email: [email protected]) Received December 15, 1999

Abstract Vegetative growth of Chinese cabbage undergoes the four successive stages which are characterized with the definite types of juvenile, rosette, folding and head leaves. From shoot tips of Chinese cabbage at early folding stage, we constructed a cDNA library and screened the differentially expressed cDNA clones using the cDNAs derived from developing folding leaves and rosette leaves as probes. One complete length of cDNA clone is designated as BcpLH. Computer alignment matched BcpLH to the domains of double-stranded RNA binding (DBRM) and the homologous regions were recognized between BcpLH and human and mouse double-stranded RNA-binding protein TRBP. PCR expression analysis shows that during vegetative growth BcpLH gene was expressed preferentially in folding leaves at folding stage. Transcripts of BcpLH gene were increased when plants were sprayed with IAA. It is deduced that BcpLH gene may be related to initiation of folding leaf and leafy head and induced by auxin in the aspect of transcriptional expression. Keywords:

Chinese cabbage, gene expression, RNA binding protein, auxin, leafy head.

Chinese cabbage (Brassica campestris ssp. pekinensis), belonging to the genus Brassica, is one of the most important vegetable crops in China and the Fareast. Under the condition of cultivation, Chinese cabbage goes through a long period of vegetative growth that may be divided into four stages: seedling stage, rosette stage, folding stage and heading stage, characterized respectively with the apparent morphological markers of leaf[1]. At seedling stage, primary and juvenile leaves are differentiated and grow; at rosette stage, plant forms rosette by extending rosette leaves; upon entering the folding stage, the folding leaves are differentiated; at heading stage, the heading leaves become more and bigger. Juvenile leaves, rosette leaves, folding leaves and heading leaves are four types of leaves with difference in shape, size, color and physiological function. The for-

* The nucleotide sequence data reported in this paper will appear in the DDBJ/EMBL/GenBank Nucleotide Sequence Database under the accession number: Y16954.

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mation of heading leaves characterizes the end of the rosette stage and the start of heading leaf differentiation. The heading leaves are curved inward and in the half-folding state at the heading stage. Then they cluster around the leafy head. Of course, there is no strict division among seedling stage, rosette stage, folding stage and heading stage, and the number of leaves of each type varies with cultivars and growth conditions[2]. The leafy head, composed of whorled leaves, is one kind of typical edible organs of morphological transformation. In Chinese cabbage, many elements influence the initiation and developmental process of leafy head, which are promoted by the uneven distribution of auxin in leaves, lower temperature and bigger day-night temperature difference, weak light and short daylength as well as enough carbohydrate nutrition[2]. Clearly, initiation of leafy head is a complicated biological process and morphological reaction induced by environmental factors. During this process, the expression and regulation of genes are crucial for plant to sense outer signal and to switch the developmental activities of plant. Little is known about function of gene in regulation of leafy head development for a long time, and then the understanding of morphogenetic process of leafy head is largely limited because the result of many physiological experiments could not be explained and verified perfectly. In order to evaluate correctly the physiological mechanism of the differentiation and development of leafy head, the related genes must be isolated and their structure features and expression pattern designated. In this way, biochemical chain arranged by gene expression induced by hormones and environmental components will be opened and the relationship between environment, gene and morphogenesis established which is also useful for understanding the complicated mechanism of leafy head formation and organogenesis of other plant species. From 1993, we have tried to isolate and clone the gene related to leafy head development, and to find the corresponding genes controlling the process of differentiation and development of leafy head. This work could offer more evidence for regulation of development process of leafy head and creation of new varieties of high yield and high quality with gene engineering. 1

Materials and methods

1.1

Plant materials The inbred line Da1-12 of Chinese cabbage was chosen for the experiment. It is the variety of early maturity, and its growth and leafy head development is sensitive to environmental conditions such as temperature and light. The seeds of this inbred line were selected carefully and sowed in the field of farm station of Shanghai Institute of Plant Physiology, Chinese Academy of Sciences at the same time of normal sowing in Shanghai (August 20, 1994). The seedlings were transplanted into the field 30 d after sowing. At the early folding stage, plants with 12—14 expanded leaves were marked for sampling on October 13. The shoot tips of 5 mm in length were harvested with 5—8 developing leaves from some plants while the developing leaves of shorter than 5 mm were cut from the other plants. Before this, the developing leaves of shorter than 5 mm were cut from the plants with 8—10 expanded leaves at early rosette stage on October 5. Shoot

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tips and developing leaves were collected and stored in liquid nitrogen for mRNA extraction and isolation and construction of cDNA library as well as preparation of isotope probes. 1.2

Construction of cDNA library and differential screening From the shoot tips of Chinese cabbage at the early folding stage, the total RNA was isolated by phenol-SDS extraction followed by lithium chloride precipitation. Enriched poly(A)+ RNA was isolated using oligo(dT) cellulose column ( Pharmacia, Sweden ), and cDNA synthesis was performed using Universal RiboClone cDNA Synthesis System ( Promega ) following the manufacturer’s protocol. The cDNA was ligated with EcoR I adaptor in EcoR I-digested arms of LamdaGEM-2 and packaged into bacteriophage using Packagene Lamda DNA Packaging System ( Promega ). Total RNA was isolated from the developing leaves of either early rosette stage or early folding stage employing guanidinium isothiocyanate method[3], and the total cDNA complementary with the single strand of RNA was labeled with 32P-dCTP using random primer method (Promega). Duplicate filters were prepared from shoot tip-specific cDNA library and transferred onto nitrocellulose membrane and prehybridized for 4 h at 42℃ in prehybridization solution containing 25% formamide, 100 μg/mL denatured salmon sperm DNA, 10 μg/mL yeast RNA, 5× Denhardt’s solution, 50 mmol/L sodium phosphate (pH 6.5), 5×SSC and 0.2% SDS. The cDNA probe derived from rosette leaf or folding leaf was added to the prehybridized solution and hybridized respectively with the cDNA library for 20 h. The hybridized filter was rinsed in leaching solution containing 0.05 mol/L NaH2PO4, 0.05 mol/L Na2HPO4, 5×SSC and 25% formamide at room temperature, and once in 2×SSC/0.02% SDS at 42℃. The filters were dried and exposed to X-ray film with intensifying screens overnight. Plaques that differentially hybridized to folding leaf cDNA but not to rosette leaf cDNA were rescreened at low densities. The interested clones were rescreened and selected and the cDNA inserts were amplified by PCR using T7 and SP6 primers. PCR products were cloned in pBluescript KS+ plasmids. 1.3

DNA sequence analysis The nested clones of BcpLH gene were created with exonuclease III and S1 nuclease. The plasmid of these nested clones was alkaline-lysed and manually sequenced with Sanger method. The sequence was verified through the second sequencing on a sequencer. Homology analysis was carried out with Blast program and specific domain was searched with ProDom program.

1.4

PCR expression analysis The total RNA was isolated respectively using the above-mentioned method from the roots,

stems and leaves at different developmental stages. With Oligo(dT)12—18, the reverse transcription was set up to synthesize the first strand of cDNA. In order to make comparison of transcription products of different tissues, all aliquots of RNA samples were adjusted to be the same concentration by condensing or diluting. According to the cDNA sequence of BcpLH gene, a pair of specific

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primers were designed: 5 ′-AATGAAGTTTCCTCTGGT-3 ′(sense primer) and 5 ′ACAGCTATGCTTGGCTTGC-3′(antisense primer). RT-PCR was carried out using the adjusted cDNAs from various plant organs as templates. Before PCR experiment, attention was paid to put equal amount of cDNA sample, primers and other PCR reagents in every reaction mix. For the elimination of reaction error, two controls were arranged, one being the total RNA from different plant tissue (internal control) and the other the cDNA of BcpLH gene (positive control). Each PCR reaction mix had the volume of 50 μL including 1.25 mmol/L MgCl2, 20 pmol primer and 3 unit Taq DNA polymerase. 40 cycles were designated with 1 min denaturation at 95℃, 2 min annealing at 58℃ and 2.5 min extension at 72℃. After amplification, 20 μL PCR solution was loaded on agarose gel for electrophoresis. DNA products of different size were transferred onto the nitrocellulose filter, then Southern hybridization was performed using the above-mentioned cDNA probe of BcpLH gene. The hybridized filter was autoradiographed and photographed. 2 2.1

Results Isolation of BcpLH gene According to the knowledge about the activated transformation of shoot tip of Chinese cab-

bage at various stages, 12—14 expanded leaves were determined to be the morphological sign of transition from rosette stage to folding stage. After this, the leaves differentiated from plant got the characteristics of folding leaf. From shoot tips at early folding stage, mRNA was extracted and isolated and reverse transcribed into cDNA. In this way a cDNA library specific for tissue of shoot tip at folding stage was constructed with titer of 5.4×105. Meanwhile, mRNAs were isolated from developing leaves of either rosette stage or folding stage, and both cDNA probes were synthesized with labeled cDNA of rosette and folding stages after reverse transcription. When the library was hybridized to these two cDNA probes, totally 9 clones with differential performance were selected, and three of them that gave strong signals with folding leaf cDNA probe and but no signal with rosette cDNA probe were retained through rescreening. Lamda DNA of the phages from these three clones were amplified as templates using T7 and SP6 primers, and three PCR products of different length were defined and cloned respectively in pBluescript plasmids. The inserts were digested with restriction enzymes for the construction of physical maps of DNA fragment. Southern blotting showed that these three inserts were from the cDNA library specific for shoot tip of Chinese cabbage at the early folding stage. One cDNA clone of 1.1 kb was sequenced mainly. After sequence deletion, subcloning and sequencing, it was found to have a long Poly(A)+ tail at 3′ terminus, a typical cap structure of mRNA at 5′terminus. It is deduced that the cDNA insert was complete in length and designated as BcpLH (fig. 1). The first ATG in the sequence was determined to be translational start codon. The nucleotide sequence surrounding the initiation codon, ATAATGA, is consistent with the proposed eukaryotic translation initiation consensus sequence of

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(A/G)NNATGg[4]. Meanwhile an in-frame stop codon, TGA, was found 6 nucleotides upstream from the first ATG initiation codon.

Fig. 1. cDNA and deduced amino acid sequence of BcpLH gene. Start codon and stop codon are boxed and underlined respectively, and two dsRNA binding domains are shadowed.

To depict the copy number of BcpLH gene in Chinese cabbage genome, Southern blotting of genomic DNA was employed. Under highly stringent condition (58℃), two hybridized bands of 8.0 and 4.8 kb were clearly seen when Hind III fragments were probed with cDNA of BcpLH gene,

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and one band of 5.8 kb in the case of EcoR I fragments and another band of 4.5 kb in BamH I fragments (fig. 2). Under less stringent conditions (50℃), more than 5 bands were recognized when Hind III, EcoR I or BamH I fragments were experimented separately. It is shown that there are several fragments homologous to BcpLH gene in genome of Chinese cabbage or some proteins coded by these genes embracing RNA binding domains. Therefore more Southern hybridization is planned to perform with the isotope probe derived from the DNA fragments excluded from RNA binding domains. 2.2

The structures of BcpLH gene The whole length of BcpLH cDNA is 1 092 bp, in which

there are 57 bp 5′untranslated region and 160 bp 3′UTR (fig.1).

Fig. 2. Southern blots of BcpLH gene in the genome of Chinese cabbage. Genomic DNA was digested with Hind III (H), EcoR I (E) or BamH I (B) and electrophoresed in 0.8% agarose gel. The DNA was transferred onto nitrocellulose membrane and hybridized with BcpLH cDNA probe labeled with 32 P by random priming. The DNA size is shown on the left side.

The deduced amino acid sequence of BcpLH is 274 residues. After registration in GenBank (EMBL/DDBJ/GenBAK Y16954), BcpLH gene was aligned. It was not found to have the known genes homologous in plant kingdom. Using the ProDom program which is specially used for domain search and analysis, three fragments of BcpLH were determined to be identical to double-stranded RNA binding domains (DRBM) that is structural feature of some RNA-binding proteins (fig. 3). In all RNA bind-

ing proteins characterized with DRBM, higher homology was found between the DBRM domain of Chinese cabbage BcpLH and DRBM domains of animal proteins such as human TRBP[5], mouse TRBP (Prbp)[6], fruitfly staufen[7], Xenopus TRBP[8] as well as rat DRADA[9], human DRADA[10]. Among them, the first DRBM domain and the second DRBM domain between proteins of BcpLH and human TRBP are as high as 59% and 51% respectively. Furthermore, the number of amino acid residues between two DRBM domains is almost the same, and higher homology exists between the upstream fragments of

Fig. 3. Conserved features within dsRNA binding domains of BcpLH and other dsRNA binding proteins. A comparison of BcpLH gene with human DRADA (DSRA), rat DRADA, human TRBP, Xenopus TRBP, Drosophila Staufen and human KP68. Amino acids numbers are according to the database. Amino acids are grouped by the method of Gatignol[5]. Capital letters, standard amino acid abbreviation; h, hydrophobic residues; p, polar residue; x, unassigned.

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two DRBM. 2.3

Expression pattern of BcpLH gene The temporal and spatial expression pattern was studied through the reverse transcription of mRNA isolated from various tissues at different developmental stages by following PCR analysis of the cDNAs. The roots, stems, leaves and shoot tips of Chinese cabbage are harvested at seedling stage, rosette stage, folding stage and heading stage, from which the Fig. 4. PCR expression analysis of BcpLH gene in tissues of total RNAs were isolated. The amount and Chinese cabbage. (a) Gene expression in leaves of different developmental stages. SL, Developing leaf at seedling stage; RL, purity of total RNA are varied with the sam- developing leaf at rosette stage; FF, developing leaf at folding (b) Gene expression of heading stage. OF, Outer leaf; FL, ples from different tissues and organs. When stage. folding leaf, HL, heading leaf. rRNA in the lower part of the the content of total RNA is the same in all the figure is used as control to show the equal RNA content of each sample. samples, the PCR products are proportional to corresponding mRNAs and then expression levels could be comparable between different tissues or organs. For this reason, we condensed or diluted the solutions of the total RNA derived from various tissues to make them basically contain the same amount of RNA. During the period of PCR amplification, the reactions of one group of samples were carried out under the same time and the same conditions. The primers used for amplification were in sec. 1.4. After electrophoresis on agarose gel, PCR products were transferred onto nitrocellulose membrane and hybridized with labeled cDNA of BcpLH gene. Temporal and spatial expression of BcpLH gene was found during vegetative growth (fig. 4). At seedling stage, expression of BcpLH gene was not detected in root, stem and leaf. Upon entering the rosette stage, the expression level was low in developing leaves. At folding stage, transcripts of BcpLH were significantly high in folding leaves. When plants developed into heading stage, the lowering expression was detected in folding Fig. 5. Effects of auxin treatment on transcription expression of Chinese cabbage BcpLH gene. 2 mg/L IAA was sprayed onto the dorsal surface of leaves at specific leaves, whereas no expression stages from which total RNA was isolated and hybridized with cDNA probe of was seen in root. It is apparent BcpLH gene. (a) Induction of gene expression in leaves at different stages. R, Rosette leaf at rosette stage; F, folding leaf at folding stage; H, folding leaf at head- that developing folding leaf is ing stage; C, positive control. (b) Transcripts of BcpLH gene varied with time proceeding after IAA treatment on folding leaf. Time (h) for sampling of leaves the major organ for preferential sprayed by auxin is indicated by 0, 2, 4 and 8. expression of BcpLH.

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2.4

Auxin and expression of BcpLH At rosette stage of Chinese cabbage, 2 mg/L IAA was sprayed onto the dorsal surface of rosette leaves, where total RNA was isolated. When Northern hybridization was performed with cDNA probe of BcpLH gene, the RNA aliquot gave the weak signal (fig. 5). On the contrary, when folding leaves and rosette leaves at folding stage were treated with IAA in the same way, the significant hybridization signal was seen on the lane of folding leaves. Apparently, IAA may induce the expression of BcpLH gene, and such induction function is significant in folding leaves and weak in rosette leaves. Meanwhile, the expression level increased with the process of time during 2—8 hours after treatment of IAA on the leaves sampled at time interval. 3

Discussion

The modification of plant organ is designated as the morphological and physiological transformation of organ[11]. Rosette leaf and heading leaf of Chinese cabbage are two types of leaf while the former takes the function of photosynthesis and the latter has the function of accumulating and storing nutrients such as carbohydrate substance[2]. Folding leaf is the intermediate form between rosette leaf and heading leaf that is similar to either rosette leaf or heading leaf in some aspect of morphology and physiology. The regular change of leaf form at different stages is controlled by shoot apical meristem whose functional transition in physiology and morphology is accompanied. BcpLH gene is preferentially expressed in the folding leaf at folding stage, suggesting that the gene plays a very important role in the regulation of morphogenesis of folding leaf, and its structure feature displays a novel regulatory pathway of morphogenesis control. RNA-binding protein is an important developmental regulator[12] and has different motifs in structure such as arginine-rich motif, glycine-rich motif or zinc finger motif. Structural analysis of BcpLH gene presented the fragments homologous to the double-stranded RNA binding domain of many RNA binding proteins which function in the developmental process by the modification of the RNA editing, polyadenylation, signal recognition or nuclear targeting. Besides the homology in double-stranded RNA binding domain, human and mouse TRBP shared a higher homology in the sequence flanking the RNA-binding protein with BcpLH. Therefore it is deduced that BcpLH gene may encode a plant RNA binding protein. Human TRBP can bind to HIV I virus TAR sequence and act as an inhibitor of the interferon-induced protein kinase, PKR[13]. Mouse TRBP can bind the untranslated region of the protamine mRNA and repress the translation[6]. The organ and tissue of plant are different from that of animal. How does BcpLH protein bind to dsRNA in plant? What role does BcpLH protein play during the organogenesis of plant? Nowadays, only a few of RNA-binding protein genes have been identified in plant and located only in the mitochondria and chloroplasts. BcpLH gene is a nuclear gene and its location in tissue and physiological function may be different from those of the known genes encoding RNA-binding protein. However, the results of research on animal RNA-binding protein could be applied to the plant field and then the function of the BcpLH gene understood.

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BcpLH gene is expressed in Chinese cabbage so weakly that it could not be detected with the routine Northern hybridization method. However, the transcript signal of BcpLH gene was enlarged by the RT-PCR. At the folding stage, BcpLH gene was expressed only in the folding leaf, but not in rosette leaf and root. Then it is in folding leaf that the BcpLH gene is expressed preferentially. The initiation of leafy head was characterized by bending inward of leaves and marked by differentiation of folding leaves. Although folding leaves are not the component of leafy head, they can switch morphogenesis of the leafy head. When the IAA was sprayed onto the dorsal side of folding leaf, the leaf was induced to bend inward, whereas the juvenile leaf and rosette leaf could not in the same way [2]. In our previous study, formation of leafy head was apparently earlier when auxin synthesis genes were introduced into Chinese cabbage and cabbage[14]. In the present study, the transcripts of BcpLH gene can be detected in the folding leaf, not in juvenile leaf and rosette leaf. It is clear that folding leaf is different from other kinds of leaf since it has biological competence to accept outer environmental stimuli and to transmit signals to morphological transformation. During the process, the BcpLH gene may play a regulatory role in signal trapping and transduction and then lead to the differentiation of folding leaf and initiation of leafy head in an indirect way. Acknowledgements We thank Prof. Xu Zhihong for valuable comments and Dr Lu Yuping for the technical method in constructs of cDNA library.

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