Plant Molecular Biology 19: 239-249, 1992. © 1992 Kluwer Academic Publishers. Printed in Belgium.
239
Isolation and characterization of a eDNA that encodes ECP31, an embryogenic-cell protein from carrot Tomohiro Kiyosue 1, Kazuko Yamaguchi-Shinozaki 2, Kazuo Shinozaki 2, Katsumi Higashi 1, Shinobu Satoh 1, Hiroshi Kamada 1 and Hiroshi Harada 1 l lnstitute of Biological Sciences, University of Tsukuba, Tsukuba-shi, Ibaraki, 305 Japan; 2plant Molecular Biology Laboratory, The Institute of Physical and Chemical Research (RIKEN), 3-1-1 Koyadai, Tsukuba-shi, Ibaraki, 305 Japan Received 20 August 1991; accepted in revised form 14 January 1992
Key words: ABA, Daucus carota, ECP31, gene expression, LEA clone, somatic embryogenesis
Abstract
A full-length eDNA for ECP31, an embryogenic cell protein from carrot (Daucus carota L.) with a Mr of 31000 (Kiyosue T, Satoh S, Kamada H, Harada H (1991) Plant Physiol 95: 1077-1083), was isolated from a eDNA library prepared from embryogenic cells using PCR-amplified DNA as a probe. The genomic Southern blot analysis revealed that there are two or three genes for ECP31 in the carrot gehome. The transcripts of ECP31 accumulated in the peripheral regions of clusters of embryogenic cells and disappeared in the course of somatic embryogenesis that was induced by transfer of the embryogenie cells to auxin-free media. The cDNA encodes a polypeptide of 256 amino acids, and the calculated molecular weight of this polypeptide is 26 111. The deduced amino acid sequence shows a high degree (62.2~o) of similarity to that of a protein that is abundant during late embryogenesis of cotton (LEA D34; Baker JC, Steele C, Dure III (1988) Plant Mol Biol 11: 227-291). The mRNAs for ECP31 started to accumulate in zygotic embryos at a late stage ofembryogenesis but were undetectable in mature embryos within 24 h after imbibition of seeds. In dry fruits (seeds), the transcripts were detected only in zygotic embryos by in situ hybridization. The level of ECP31 transcripts increased after treatment with abscisic acid (ABA) in torpedo-shaped somatic embryos but not in seven-day-old seedlings. These results suggest that both embryo-specific factor(s) and ABA are involved in the expression of the gene for ECP31.
Introduction
Characterization ofgene expression during developmental processes is a necessary step towards an understanding of developmental mechanisms.
Since formation of carrot somatic embryos can be readily induced by the transfer of embryogenic cells that have been cultured in auxin-containing medium to auxin-free medium [19, 20], somatic embryogenesis in carrot has provided us with a
The nucleotide sequence data reported will appear in the EMBL, GenBank and DDBJ Nucleotide Sequence Databases under the accession number X60593.
240 good system with which to investigate gene expression during early development in plants, and specific genes have been found to be expressed at certain stages of embryogenesis [1, 5,6, 8, 19, 23-26]. ECP31 is a carrot embryogenic-cell protein with a relative mass of 31000, which accumulates preferentially in clusters of embryogenic cells and disappears during the development of somatic embryos. The appearance of ECP31 in hypocotyl segments cultured on MS semisolid media that contained 2,4-D coincided with formation of embryogenic callus [ 13]. From these results, we postulated that ECP31 has an important role in the induction and/or maintenance of embryogenic competence. Recently, we reported the partial amino acid sequence of ECP31 and demonstrated that four of the five internal sequences determined were 70-807o homologous to sequences in the lateembryogenesis-abundant (LEA) protein D34 of cotton [ 14]. In the present report we describe the isolation of a full-length cDNA for ECP31 and an analysis of the expression of the corresponding gene during somatic and zygotic embryogenesis of carrot with the cDNA as a probe. Materials and methods
Plant materials and cell culture Carrot (Daucus carota L. cv. US-Harumakigosun) seeds were sown on vermiculite beds and grown at 25 °C under a 16 h light/8 h dark cycle (6.5 × 10-23 E m -2 s-i). Mature plants (D. carota L. cv. Kurodagosun) were grown in the field of Takii Co., Ltd. (Ibaraki, Japan). Clusters of embryogenic carrot cells (D. carota L. cv. USHarumakigosun) were obtained from hypocotyl segments of seven-day-old seedlings which were cultured on Murashige and Skoog's (MS) semisolid medium [18] with 1 mg/1 2,4-D, and they were maintained by transfer to fresh MS liquid medium supplemented with 1 mg/1 2,4-D every two weeks. Somatic embryos were induced by the transfer of embryogenic cells to auxin-free MS medium as described by Satoh et al. [20].
Treatments with abscisic acid (ABA) ABA was added to the medium in which torpedoshaped somatic embryos were cultured at a final concentration of 3.7 x 10 -6 M and the embryos were incubated for 1 or 24 h. Seven-day-old carrot seedlings, growing on vermiculite beds, were sprayed with a 10-4M solution of ABA and incubated for 1, 4, 8 or 24 h at 25 °C under dim light.
Isolation of RNA from carrot cells In order to construct a cDNA library, total RNA was isolated from clusters of embryogenic cells (14.5 g), which had been cultured in medium supplemented with 2,4-D, by guanidine thiocyanate method [15], and poly(A) + RNA was isolated from the RNA by two cycles of column chromatography on oligo(dT)-cellulose. For northern blot analysis, RNA was isolated from various cells by the phenol/SDS method [2].
Isolation of genomic DNA from carrot seedlings Carrot genomic D N A was extracted from 90 g of three-week-old seedlings by the protocol of Maniatis et al. [ 15].
Preparation of a cDNA library Poly(A) + RNA, prepared from clusters of embryogenic cells, was used to construct a cDNA library in lambda gtl 1 by use of the cDNA Synthesis System Plus and the cloning system from Amersham (Aylesbury, UK). The library was amplified and used for further experiments.
Amplification of target DNA by the polymerase chain reaction (PCR) The amplification of the specific D N A was performed by the polymerase chain reaction (PCR),
241 using the Gene Amp DNA Amplification Reagent Kit (Perkin Elmer Cetus, Norwalk, CT). The primers for PCR consisted of the forward primer, 5 ' -GGIAA(A/G)ACICA(A/G)AA(A/G)GGIG GICC-3', derived from the amino acid sequence GKTQKGGP, and the reverse primer, 5'-GCI ACICCIGG (A/G) TAIGTCATCAT-3 ', derived from the amino acid sequence MMTYPGGVA, which were reported previously [14]. The template for PCR was 100 ng of the DNA from the cDNA library from the clusters of embryogenic cells and either one of the two primers was added at 100 pmol. The denaturing and annealing processes during the reaction were performed at 94 °C for 1 min and 37 °C for 2 min, respectively, and the synthesis of DNA from the primers was catalyzed by the thermoresistant Taq I polymerase at 72 °C for 3 min.
Screening and subcloning of the cDNA library Recombinant phages were plated at high density (50000 plaques per 154 cm 2 dish), transferred onto nylon filters (Dupont, Boston, MA) and screened by hybridization with [32p]-labeled 600 bp PCR fragments using a Random Primed DNA Labeling Kit (Boehringer Mannheim, Mannheim, FRG). Hybridization was performed according to the instructions from the manufacturers of the nylon filters. Lambda DNA was isolated by ultracentrifugation on a CsC12 gradient [15]. The cloned cDNA was isolated from Eco RIdigested lambda DNA, separated on a 0.7 ~o lowmelting-temperature agarose gel and electroeluted from gel slices in an electroeluter (IBI, New Haven, CT). The cDNA was ligated into pBluescript SK- (Stratagene, La Jolla, CA) and M13mpl8 or M13mpl9 phage, and Escherichia coli strains XL-1 and JM109 served as hosts for the plasmid and phage, respectively.
sequenced by the dideoxynucleotide chain-termination method with Sequenase (USB, Cleveland, OH) in accordance with the manufacturer's protocols.
Hybridization of RNA Total RNA (20 ~g) from carrot tissues or cultured cells was denatured with 2.15 M formaldehyde and 50~o (w/v) formamide and then fractionated by electrophoresis in a 1.2~o (w/v) agarose gel that contained 2.2 M formaldehyde [14]. The RNA was subsequently transferred to a nitrocellulose membrane in 20x SSC (1 x SSC -- 0.15 M NaC1, 0.015 M trisodium citrate, pH 7.0) as transfer buffer. The transferred RNAs were hybridized with [32p]-labeled fragments ofcDNA, labeled with the Random Primed DNA Labeling Kit. Prehybridization, hybridization, and posthybridization steps were performed essentially as described elsewhere [15]. The fragments ofcDNA that hybridized to specific RNAs were visualized by autoradiography.
Hybridization of DNA Carrot DNA was digested with restriction endonucleases and the resulting fragments were separated according to size on a 0.7~o (w/v) agarose gel and then transferred to a nitrocellulose membrane for hybridization as described elsewhere [ 15]. The transferred DNAs were hybridized with [32p]-labeled fragments of cDNA, labeled with the Random Primed DNA Labeling Kit and the hybridized fragments were visualized by autoradiography.
In situ hybridization using digoxigenin-labeled
probes DNA sequence analysis The plasmid DNA templates and the singlestranded DNA templates from M13 phage were
Clusters of embryogenic carrot cells and dry fruits (seeds) were fixed with 4 ~o paraformaldehyde in PBS (10 mM sodium phosphate, 150 mM sodium chloride, pH 7.0) for 12 h at 4 °C. The fixed sam-
242 ples were embedded in O.C.T. compound (Miles Scientific, Naperville, IL) and frozen at -80 ° C. Sections (20 #m) were cut at -25 °C on a cryostat and transferred to glass slides, which had been coated with a 2~o solution of Vectabond reagent (Vector Laboratories, Burlingame, CA) in acetone. Prior to synthesis of RNA, the plasmids containing ECP31 cDNA were linearized with Xho I (when T3 polymerase was used) or Xha I (when T7 polymerase was used). Antisense and sense RNA probes were transcribed from T3 and T7 promoters of the recombinant pBluescript using a digoxigenin-labeling kit (Genius Kit; Boehringer Mannheim). The prehybridization treatments, the hybridization with RNA probes, the posthybridization treatments and the immunological detection were performed essentially as described in the Technical Bulletin from Boehringer Mannheim Biochemicals. After the color had developed, the sections were rinsed for 30 min with two changes of distilled water and mounted with 50~o glycerol that contained 7~o gelatin. Photographs were taken under a light microscopy, Vanox model AHBT (Olympus, Tokyo, Japan).
from the proteolytic fragments of ECP31 on a gas-phase protein sequencer [14]. The amplified 600 bp fragments of D N A were used as a probe for screening the cDNA library. A total of 300 000 phages were screened and 12 cDNA clones were isolated. Eight clones had inserts of about 1.05 kb, two had inserts of 0.8 kb and two contained a fragment of 2.7 kb. Partial sequencing from both the 5' and the 3' termini showed that there were at least two rather similar kinds of ECP31 cDNA of 1.05 kb and that the 0.8 kb fragments were truncated forms of the 1.05 kb fragments. The 2.7 kb fragments were found to be artifacts derived from combinations of ECP31 cDNAs and ribosomal DNA. A 1.05 kb fragment was subcloned into the Eco RI site of pBluescript and the resultant plasmid was designated pcECP31.
Gel-blot analysis of genomic DNA with pECP31 Genomic D N A from three-week-old carrot seedlings was digested with Hind III, Bam HI and Eco RI. Hybridization was carried out at 42 °C with [32p]-labeled pcECP31 (Fig. 1). After digestion with Hind III and with Barn HI, 3.9 kbp and
Results
Amplification by PCR of target DNA and isolation of cDNA that encoded ECP31 Poly(A) + RNA was isolated from clusters of embryogenic carrot cells and used for construction of a cDNA library with the lambda gtl 1 vector. In order to obtain a probe for screening of the c D N A library and isolation ofcDNAs for ECP31, polymerase chain reactions (PCRs) were carried out using 100 ng of the isolated DNAs from the c D N A library at an annealing temperature of 37 ° C, for 30 cycles, with the primers GGIAA(A/ G) ACICA (A/G) AA (A/G) G G I G G I C C and GCIACICCIGG (A/G) TAIGTCATCAT, derived from the amino acid sequences GKT Q K G G P and MMTYPGGVA, respectively. These sequences had been previously determined
Fig. 1. Genomic Southern blot analysis ofECP31 genes. Total DNA was digested with the restriction endonucleases indicated and probed with the [32P]-labeled cDNA of pcECP31. H, Hind III; B, Barn HI; E, Eco RI. Fragments of Hind IIIdigested lambda DNA were used as size markers.
243 4.2 kbp bands were detected, respectively, and two hybridizing bands of 6.5 kb and 15 kb were detected after digestion with Eco RI. Since at least two different but similar cDNA clones were obtained by the screening, it is likely that there are two or three genes for ECP31 within about 4 kb of carrot genomic DNA.
Accumulation of ECP31 mRNA during somatic embryogenesis
Northern blot analysis was used to determine the levels of ECP31 m R N A during somatic embryogenesis. The results are shown in Fig. 2. Clusters of embryogenic cells were found to contain a high level of ECP31 mRNA. When somatic embryogenesis was induced by transfer of the embryogenic cells to medium without 2,4-D, the level of this m R N A decreased and little of this m R N A was present at detectable levels in torpedo-shaped somatic embryos. In situ hybridization demon-
Fig. 2. Northern blot analysis of ECP31 mRNA during formation of somatic embryos. Total RNA was extracted from embryogenic cells (lane 1), somatic embryos at the heart stage, which correspond to those on the 10th day of culture after transfer of embryogenic cells to auxin-free medium (lane 2) and somatic embryos at the torpedo stage, which correspond to those on the 14th day of culture after transfer (lane 3). Twenty #g each of total RNA were separated on a 1.2% agarose gel that contained 2.2 M formaldehyde. The RNAs were subsequently blotted onto a nitrocellulose membrane and probed with [32P]-labeled cDNA from pcECP31.
strated that ECP31 transcripts were preferentially localized in the peripheral cells of the clusters of embryogenic cells (Fig. 3), where pro-embryos originated [12]. Because cell density of the outer part of the cluster is not significantly different from that of the inner part, the peripheral embryogenic cells accumulated more ECP31 transcripts than other cells of the cluster.
Analysis of the nucleotide and amino acid sequences
The 1012 bp nucleotide sequence of pcECP31 is shown, together with the deduced amino acid sequence, in Fig. 4. After the stop codon, pcECP31 has a 197 bp 3'-non-coding region which is followed by 19 adenyl residues and the sequence AATAAG which resembles the AATAAA motif 75bp upstream from the polyadenyl tail. pcECP31 contains an open reading frame of 256
Fig. 3. Localization of ECP31 mRNA in clusters of embryogenic cells by in situ hybridization. Sections of clusters of embryogenic cells were hybridized with single-stranded digoxigenin-labeled sense (A) and antisense (B) RNA probes of ECP31 cDNA.
244 i0 20 30 40 50 60 70 80 90 C~&~AG~a~ATTTACACACAAATCCTC`&gC~&~a~AATACAAAGAT~A~CCA~C~C~CCAC~AGACCC~C~G~A~CCCATCAAATAC~A M S Q Q Q P R R P Q Q E Q P I K Y G
i00 D
ii0 120 130 140 150 160 170 180 190 c~TATTT~AT~TTTCC~cA~TT~AGCTCCC~CCC~TCGCCCCc~C~AC~CCTcc~CcAT~CA~cTGcG~AG~TAT~T~CTTG~T~%GACGCAG V F D 47 S G Q L S S Q P V A P N D A S A M Q A A E N M V L G 210 220 230 240 250 260 270 280 ~GGT~TCC~CCTCTGTcAT~CA~Tc~C~c~CT~CT~TCTGCA~CGC~C~Tc~TTG~CCcGCAcGA~cACCCCcATA~CTA~T~C~ K G G P A S V M Q S A A A A N L Q R G V V G P H E G T P I A S
200 K
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290 E
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300 G
310 320 330 340 350 360 370 380 390 ~G~TCACTATTTCTG~cTGA/~%TCGCTGG~%CTCG~TCATTAccGAGGCTGTCGGA~ACAGGTGGT~GGGC~TACTTG~CCAG~GN%%TTT~ V T I S E A E I A G T R I I T E A V G G Q V V G Q Y L E P G K F K
400
410 420 430 440 450 460 470 480 490 ~ATGCC~CCC~cTGGT~T~cTCG~cA~T~ATTCGATTACTATAGGC~AG~CTTT~A~ACCACTGCCTT~Cc~cCG~AGAT~GCCTGTA~ATCAG M P S P A G V L G S D S I T I G E A L E T T A L T A G D K P V D Q
500
510 520 530 540 550 560 570 580 590 A~T~AT~CA~TGCTATAC~CA~TG.~T~AGA~C~T~CTAT~TTATCCT~T~A~TG~C~CAG~T~CTC~TCT~C~C~ATTAT~T~
600
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610 620 630 640 650 660 670 680 cTC~TACCAT~T~TT~CCA~C~CT~GCTC~TGATGTcCTT~CT~ATC~ATc~ATTAG~TT~CT~AC~C~T~AC~AGGG~AT~C R T M N V A S K T K L G D V L A D R S I R L A E D K A 710
720
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760
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690 V
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~A~A~T~TT~CA~T~A~C~TCCG~A~AT~ATGACTTAT~CT~GT~A~T~CTTCTTCCAT~CT~CTG~A~CCA~CTT~TC~ E
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810 820 830 840 850 860 870 880 890 ~ACCC~CTTTCT~TATTCCAT~T`~%AcATATCTATc~GTATTATTATGTAG~T~TGT~TTTTCAT~TACA~T~TTT~A~`~T~TA~TATcTATT D P T F ~ 910
920
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~TCATATTGTGTGTAT@--~GCTTGCGTACTA~TTGATG~AGTGCGTGTG~ACTGCTGTTTTGGTTG~TGC~GGG~TGGTTTTGGTGT~ I010 AAAAAAAAAAAA
Fig. 4. Nucleotide sequence of the cDNA ~ r ECP31 and the deduced amino acid sequence. The amino acid sequences identical to those ~ u n d in purified ECP31 are underlined. The putative polyadenylation s i s a l is boxed.
amino acids which begins with a methionine codon, and the calculated relative mass of this polypeptide is 26 111. The five sequences of amino acids that were previously determined by proteolytic digestion on membrane and subsequent analysis on a protein sequencer [14] are shown by underlines. The polypeptide has compositional bias towards Ala (16.86~) and Gly (10.20~) and it lacks Cys and Trp residues. The predicted amino acid sequence encoded by cECP31 was used in a search for homologous proteins in the SWIS S-PROT (European Molecular Biology Laboratory, Heidelberg, FRG) protein database, release 14, using the similarity search program. As a result, extensive homology (62.2~o) was found between ECP31 and lateembryogenesis-abundant (LEA) protein D34 of cotton [3] (Fig. 5).
Expression of the gene for ECP31 in plants Since the expression of the gene for LEA D34 was detected at the late stages of the formation of cotton seeds [3, 10], we were prompted to examine the expression of the gene for ECP31 during the maturation and germination of carrot seeds. ECP31 transcripts were not detected from 3 days to 23 days after anthesis but they began to be found from 28 days after anthesis (Fig. 6A). Relatively large amounts of ECP31 m R N A accumulated in dry fruits (seeds) but this m R N A became undetectable within 24 h after imbibition (Fig. 6B). The difference of the size of the hybridized mRNAs between seeds and embryogenic cells may be due to the difference of the modification of the m R N A or that embryogenic cells have electrophoresis-disturbing substances. In
245 ECP31
i' MSQQQPRRPQQ . . . . . . .
LEA D34
1" MSQGQPRRPQQPAGQGENQEPIKYGDVFNVSGELANKPIAPQDAAMMQTAETQVLGQTQK
EQPIKYGDVFDVSGQLSSQPVAPNDASAMQAAENMVLGKTQK
54'
GGPASVMQSAAAANLQRGVVGPHEGTPIASEQGVTISEAEIAGTRIITEAVGGQVVGQYL
61"
GGTAAVMQAAATRNEQVGVVGHNDITDIAGEQGVTLAETDVAGRRIITEAVAGQVVGQYV
114'
EPGKFKMPSPAGVLGSDSITIGEALETTALTAGDKPVDQSDAAAIQAAEVRASG--YAYP
121"
QATPV-MTSQVGVVLQNAITIGEALEATAKTAGDKPVDQSDAAAVQAAEVRATGSNVIIP
172'
GGVAAAAQSAADYNARTMNVASKTKLGDVLADRSIRLAEDKAVTREDAEGVVGAEVRNNP
180"
GGLAATAQSAAAHNATLDRDEEKIKLNQVLTGATAKLPADKAVTRQDAEGVVSAELRNNP
232'
EMMTYPGGVASSMAAAARLNQDPTF
240"
NVATHPGGVAASMAAAARLNENVNA
Fig 5.
C o m p a r i s o n of amino acid sequences between ECP31 and L E A D34. C o m m o n amino acids are indicated by asterisks and similar amino acids are i n d i c t e d by dots.
situ hybridization using a digoxigenin-labeled RNA probe revealed the embryo-specific accumulation of ECP31 m R N A in seeds (Fig. 7).
Regulation by ABA of the accumulation of ECP31 mRNA ABA may play a role in the regulation of expression of some classes of genes at late embryonic stages [11, 21]. To investigate the effect of ABA on the accumulation of ECP31 mRNA, RNA was extracted from torpedo-shaped somatic embryos which had been grown for 13 days without 2,4-D. Prior to the extraction, ABA (3.7 x 10-6 M) was added to the culture medium and somatic embryos were collected at 1 and 24 h after the start of this treatment. As shown in Fig. 8, little level of ECP31 transcripts was found in control somatic embryos, but the level increased during the 24 h that followed the application of ABA up to the same level in embryogenic cell clusters. Next we investigated whether or not the effect of ABA on expression of the ECP31 gene was embryo-specific. Seven-day-old carrot seedlings were sprayed with a 10-4M solution of ABA and collected at 1, 4, 8, or 24 h after this treatment. Northern blot analyses demonstrated that there was no detectable ECP31- specific m R N A in any of the ABA-treated carrot seedlings (data not shown).
Discussion
ECP31 is an intracellular protein isolated from clusters of embryogenic carrot cells, with a relative mass of 31000. It accumulates in clusters of embryogenic cells and disappears in the course of formation of somatic embryos. The appearance of ECP31 in hypocotyl segments cultured on MS semisolid medium supplemented with 2,4-D coincides with the formation of embryogenic callus. ECP31 in embryogenic callus was demonstrated to be located preferentially in the peripheral regions, in which pro-embryos are known to originate [ 12]. Given these results, we postulated previously that ECP31 performs important role(s) in the induction and/or maintenance of embryogenic competence [13]. In this report, we describe the isolation of a c D N A that encodes ECP31 and an examination of expression of the gene for ECP31 using the cDNA as a probe. First, we performed PCR using oligonucleotide primers derived from two previously determined internal sequences of amino acids in ECP31, and an amplified 600 bp fragment was used as a probe for screening the c D N A library from cultured embryogenic cells. Among 300000 recombinant phages screened, 12 clones were isolated and finally the cDNA from one clone (full-length clone) was used for further experiments. Results of genomic Southern hybridization, as well as sequence analysis of cDNA clones, dem-
246
Fig. 6. Northern blot analysis of ECP31 mRNA during development (A, upper) and germination (B, lower) of seeds. Total RNA was extracted from fruits (seeds) or seedlings of carrot. Twenty/~g of each RNA were separated on an agarose gel, blotted onto a membrane and probed with [32P]-labeled cDNA of pcECP31. A. Lane 1, fruits 3 days after anthesis (3DAF); Lane 2, 8 days; lane 3, 13 days; lane 4, 18 days; lane 5, 23 days; lane 6, 28 days after anthesis. B. Lane 1, dry fruits (seeds); lane 2, seeds 1 day after sowing; lane 3, 2 days; lane 4, 3 days; lane 5, 4 days; lane 6, 5 days; lane 7, 7 days. Twenty #g of RNA from clusters of embryogenic (EG) cells were indicated as a positive control (lane 8).
onstrated that D. carota has at least two related genes that encode ECP31. This result supports the result that ECP31 can be resolved into two polypeptides, each with a relative mass of 31000, by two-dimensional PAGE (unpublished results). A large amount of ECP31-specific mRNA was found in embryogenic cell cultures but the level of this m R N A decreased and finally it became undetectable at the torpedo stage, during the course of the development of somatic embryos. In situ
hybridization demonstrated that transcripts of the gene for ECP31 were preferentially situated in the peripheral region s of cluster s of embryogenic cells. These results were essentially the same as those obtained with an ECP31-specific antibody [13]. Thus, not only the accumulation of ECP31 but also the expression of its gone is correlated with the embryogenic competence of carrot cells. The DNA complementary to ECP31-specific mRNA consisted of 1015 bp and the amino acid sequence of the protein was deduced to be that of a polypeptide with a Mr of 26 111. The reason for the difference in size from that obtained by SDSPAGE (31000) may be related to some modifications, such as kinations (ECP31 has six putative casein kinase II phosphorylation sites and two putative protein kinase C phosphorylation sites), or to the nature of the protein. The search of the database revealed that ECP31 exhibits significant homology at the nucleotide level (42.5 ~o) and at the protein level (62.2~o) to the LEA D34 clone [3]. LEA mRNAs, which were isolated initially from cotton cotyledons [3, 10], are expressed of high levels at late embryonic stages and their levels decrease during the course of germination of seeds. Recent studies demonstrated that not only cotton but also other plant species, such as carrot, barley, rice, rape and wheat, have LEA mRNAs [9, 21]. From the conservation of various sequences, these clones have been classified into three groups: group 1, wheat Em and cotton D19; group 2, rice rabl6 (formerly rab21), cotton D l l and barley dehydrin; group 3, cotton D7, carrot Dc3, carrot Dc8, barley pHVal and rape pLEA 76 [9]. Our present results indicate the existence of an additional fourth group of Lea clones, namely cotton D34 and carrot pcECP31, in the plant kingdom. Some LEA proteins have been predicted to protect plant cells from water stress, on the basis of the patterns of their expression and their extensive hydrophilic characteristics [9]. However, ECP31 and D34 do not have such hydrophilic characteristics. This difference implies that they are probably functionally different from the other LEA proteins. The expression of the genes for cotton LEAs,
247
hybridization of the digoxigenin-labeledECP31 RNA probe in carrot seeds. Section from dry carrot fruits (seeds) were hybridized with single-stranded digoxigenin-labeled sense (A) and antisense (B) RNA probes of ECP31 cDNA. EM, embryo; EN, endosperm; OD, oil duct; P, pericarp. Bar = 100 #m. Dark purple signal found in the zygotic embryo indicates the existence of ECP31-mRNAs. Black color of the cell walls of endosperm and pericarp is due to the focus.
Fig. 7. In situ
including D34, was reported to be highly induced by treatment with ABA in premature zygotic embryos [ 10], but their organ specificity has not yet been reported; their homologues, such as rabl6, barley dehydrin and wheat Era, are induced by ABA without organ specificity [7, 16, 17, 21, 27]. The northern analysis of m R N A for ECP31 showed that the m R N A accumulated at late stages of embryogenesis, as in the case of cotton LEAs and their homologues, and the m R N A was located only in zygotic embryos (Fig. 8). Moreover, as shown in Fig. 9, treatment with ABA highly enhanced expres sion of the gene for ECP31 in torpedo-shaped somatic embryos, as reported
in cotton zygotic immature embryos, but not in carrot seedlings. This embryo-specific expression is unique among LEA m R N A s examined to date, and our present results suggest that both embryospecific factor(s) and ABA are involved in the expression of the gene for ECP31. The high level of expression of the gene for ECP31 in embryogenic cells implies the existence of both embryospecific factor(s) and of a high level of endogenous ABA in those cells. In preliminary experiments, endogenous level of ABA in embryogenic cells was estimated to be higher than that in somatic embryos, and was comparable to that in mature carrot seeds. Thus, the characteristic of
248
References
Fig. 8. Effect of ABA on expression of the gene for ECP31 in carrot somatic embryos. Total RNA was extracted from somatic embryos, fractionated on an agarose gel, blotted onto a membrane and probed with [32p]-labeled cDNA ofpcECP31. Twenty/~g of total RNA were loaded in each lane. Lane 1, control, 15 days after the transfer to medium without 2,4-D. ABA was added to the medium and samples were collected after 1 h (lane 2) and 24 h (lane 3).
E C P 3 1 a n d o t h e r c a r r o t e m b r y o n i c genes, s u c h as D c 3 a n d D c 8 , b e i n g e x p r e s s e d a l r e a d y at t h e e m b r y o g e n i c cell c l u s t e r stage, while t h e y o n l y a p p e a r m u c h l a t e r in z y g o t i c d e v e l o p m e n t , m a y b e d u e to t h e high level o f e n d o g e n o u s A B A in t h e e m b r y o g e n i c cell clusters.
Acknowledgements T h i s r e s e a r c h w a s s u p p o r t e d in p a r t b y G r a n t s i n - A i d for S p e c i a l R e s e a r c h o n P r i o r i t y A r e a s ( P r o j e c t 02242102, C e l l u l a r a n d M o l e c u l a r B a s i s for R e p r o d u c t i v e P r o c e s s e s in P l a n t s ) f r o m t h e Ministry of Education, Science and Culture, Japan, and by Special Coordination Funds of the Science and Technology Agency of the Japanese Government.
i. Aleith F, Richter G: Gene expression during induction of somatic embryogenesis in carrot cell suspensions. Planta 183:17-24 (1990). 2. Ausubel FM, Brent R, Kingston RE, Moore DD, Seidman JG, Smith JA, Struhl K: In: Current Protocols in Molecular Biology, Chapter 4. Greene Publishing Associates/Wiley-Interscience, New York (1989). 3. Baker JC, Steele C, Dure III: Sequence and characterization of 6 Lea proteins and their genes from cotton. Plant Mol Biol 11:227-291 (1988). 4. Borkird C, Sung ZR: Isolation and characterization of ABA-insensitive cell lines of carrot. Plant Physiol 84: 1001-1006 (1987). 5. Borkird C, Choi J, Jin Z, Franz G, Hatzopoulos P, Chorneau R, Bonas U, Pelegri F, Sung ZR: Developmental regulation of embryonic genes in plants. Proc Natl Acad Sci USA 85:6399-6403 (1988). 6. Choi JH, Liu LS, Borkird C, Sung ZR: Cloning of genes developmentally regulated during plant embryogenesis. Proc Natl Acad Sci USA 84:1906-1910 (1987). 7. Close TJ, Kortt AA, Chandler PM: A cDNA-based comparison of dehydration-induced proteins (dehydrins) in barley and corn. Plant Mol Biol 13:95-108 (1989). 8. de Vries S, Booij H, Meyerink P, Huisman G, van Spanje M, Wilde D, Thomas TL, van Kammen A: Acquisition of embryogenic potential in carrot cell suspension cultures. Planta 176:194-204 (1988). 9. Dure III L, Crouch M, Harada J, Ho T-H D, Mundy J, Quatrano R, Thomas T, Sung ZR: Common amino acid sequence domains among the LEA proteins of higher plants. Plant Mol Biol 12:475-486 (1989). 10. Galau GA, Hughes DW, Dure III L: Abscisic acid induction of cloned cotton late embryogenesis-abundant (Lea) mRNAs. Plant Mol Biol 7:155-170 (1986). 11. Goldberg RB, Barker SJ, Perez-Grau L: Regulation of gene expression during plant embryogenesis. Cell 56: 149-160 (1989). 12. Halperin W: Alternative morphogenetic events in cell suspensions. Am J Bot 53:443-453 (1966). 13. Kiyosue T, Satoh S, Kamada H, Harada H: Purification and immunohistochemical detection of an embryogenic cell protein in carrot. Plant Physiol 95:1077-1083 (1991). 14. Kiyosue T, Nakayama J, Satoh S, Isogai A, Suzuki Y, Kamada H, Harada H: Partial amino acid sequence of ECP31, a carrot embryogenic cell protein, and enhancement of its accumulation by abscisic acid in somatic embryos. Planta 186:337-342 (1992). 15. Maniatis T, Fritsch EF, Sambrook J: Molecular Cloning; A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1982). 16. Marcotte WR, Russell SH, Quatrano RS: Abscisic acidresponsive sequences from the Em gene of wheat. Plant Cell 1:969-976 (1989). 17. Mundy J, Chua NH: Abscisic acid and water-stress in-
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duce the expression of a novel rice gene. EMBO J 7: 2279-2286 (1988). Murashige T, Skoog F: A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15:473-497 (1962). Nomura K, Komamine A: Molecular mechanisms of somatic embryogenesis. Oxf Surv Plant Mol Cell Biol 3: 456-466 (1986). Satoh S, Kamada H, Harada H, Fujii T: Auxin-controlled glycoprotein release into the medium of embryogenic carrot cells. Plant Physiol 81:931-933 (1986). Skriver K, Mundy J: Gene expression in response to abscisic acid and osmotic stress. Plant Cell 2:503-512 (1990). Steward FC, Mapes MO, Smith J: Growth and organized development of cultured cells. I. Growth and division of freely suspended cells. Am J Bot 45:693-703 (1958).
23. Sung ZR, Feinberg A, Chorneau R, Borkird C, Turner I, Smith J, Terzi M, LoSchiavo F, Giuliano G, Pitto L, Nuti-Ronchi V: Developmental biology of embryogenesis from carrot cultures. Plant Mol Biol Rep 2:3-14 (1984). 24. Thomas TL, Wilde HD: Analysis of gene expression in carrot somatic embryos. In: Terzi J, Pitto L, Sung ZR (eds) Somatic Embryogenesis, pp. 77-85. IPRA, Rome (1985). 25. Thomas TL, Wilde HD: Analysis of carrot somatic embryo gene expression programs. Proceedings of the VIInternationalCongress on Plant Cell and Tissue Culture, pp. 83-93 Alan R. Liss, New York (1987). 26. Wilde HD, Nelson WS, Booij H, de Vries SC, Thomas TL: Gene-expression programs in embryogenic and nonembryogenic carrot cultures. Planta 176:205-211 (1988). 27. Yamaguchi-Shinozaki K, Mino M, Mundy J, Chua NH: Analysis of an ABA-responsive rice gene promoter in transgenic tobacco. Plant Mol Biol 15:905-912 (1990).
239
Isolation and characterization of a eDNA that encodes ECP31, an embryogenic-cell protein from carrot Tomohiro Kiyosue 1, Kazuko Yamaguchi-Shinozaki 2, Kazuo Shinozaki 2, Katsumi Higashi 1, Shinobu Satoh 1, Hiroshi Kamada 1 and Hiroshi Harada 1 l lnstitute of Biological Sciences, University of Tsukuba, Tsukuba-shi, Ibaraki, 305 Japan; 2plant Molecular Biology Laboratory, The Institute of Physical and Chemical Research (RIKEN), 3-1-1 Koyadai, Tsukuba-shi, Ibaraki, 305 Japan Received 20 August 1991; accepted in revised form 14 January 1992
Key words: ABA, Daucus carota, ECP31, gene expression, LEA clone, somatic embryogenesis
Abstract
A full-length eDNA for ECP31, an embryogenic cell protein from carrot (Daucus carota L.) with a Mr of 31000 (Kiyosue T, Satoh S, Kamada H, Harada H (1991) Plant Physiol 95: 1077-1083), was isolated from a eDNA library prepared from embryogenic cells using PCR-amplified DNA as a probe. The genomic Southern blot analysis revealed that there are two or three genes for ECP31 in the carrot gehome. The transcripts of ECP31 accumulated in the peripheral regions of clusters of embryogenic cells and disappeared in the course of somatic embryogenesis that was induced by transfer of the embryogenie cells to auxin-free media. The cDNA encodes a polypeptide of 256 amino acids, and the calculated molecular weight of this polypeptide is 26 111. The deduced amino acid sequence shows a high degree (62.2~o) of similarity to that of a protein that is abundant during late embryogenesis of cotton (LEA D34; Baker JC, Steele C, Dure III (1988) Plant Mol Biol 11: 227-291). The mRNAs for ECP31 started to accumulate in zygotic embryos at a late stage ofembryogenesis but were undetectable in mature embryos within 24 h after imbibition of seeds. In dry fruits (seeds), the transcripts were detected only in zygotic embryos by in situ hybridization. The level of ECP31 transcripts increased after treatment with abscisic acid (ABA) in torpedo-shaped somatic embryos but not in seven-day-old seedlings. These results suggest that both embryo-specific factor(s) and ABA are involved in the expression of the gene for ECP31.
Introduction
Characterization ofgene expression during developmental processes is a necessary step towards an understanding of developmental mechanisms.
Since formation of carrot somatic embryos can be readily induced by the transfer of embryogenic cells that have been cultured in auxin-containing medium to auxin-free medium [19, 20], somatic embryogenesis in carrot has provided us with a
The nucleotide sequence data reported will appear in the EMBL, GenBank and DDBJ Nucleotide Sequence Databases under the accession number X60593.
240 good system with which to investigate gene expression during early development in plants, and specific genes have been found to be expressed at certain stages of embryogenesis [1, 5,6, 8, 19, 23-26]. ECP31 is a carrot embryogenic-cell protein with a relative mass of 31000, which accumulates preferentially in clusters of embryogenic cells and disappears during the development of somatic embryos. The appearance of ECP31 in hypocotyl segments cultured on MS semisolid media that contained 2,4-D coincided with formation of embryogenic callus [ 13]. From these results, we postulated that ECP31 has an important role in the induction and/or maintenance of embryogenic competence. Recently, we reported the partial amino acid sequence of ECP31 and demonstrated that four of the five internal sequences determined were 70-807o homologous to sequences in the lateembryogenesis-abundant (LEA) protein D34 of cotton [ 14]. In the present report we describe the isolation of a full-length cDNA for ECP31 and an analysis of the expression of the corresponding gene during somatic and zygotic embryogenesis of carrot with the cDNA as a probe. Materials and methods
Plant materials and cell culture Carrot (Daucus carota L. cv. US-Harumakigosun) seeds were sown on vermiculite beds and grown at 25 °C under a 16 h light/8 h dark cycle (6.5 × 10-23 E m -2 s-i). Mature plants (D. carota L. cv. Kurodagosun) were grown in the field of Takii Co., Ltd. (Ibaraki, Japan). Clusters of embryogenic carrot cells (D. carota L. cv. USHarumakigosun) were obtained from hypocotyl segments of seven-day-old seedlings which were cultured on Murashige and Skoog's (MS) semisolid medium [18] with 1 mg/1 2,4-D, and they were maintained by transfer to fresh MS liquid medium supplemented with 1 mg/1 2,4-D every two weeks. Somatic embryos were induced by the transfer of embryogenic cells to auxin-free MS medium as described by Satoh et al. [20].
Treatments with abscisic acid (ABA) ABA was added to the medium in which torpedoshaped somatic embryos were cultured at a final concentration of 3.7 x 10 -6 M and the embryos were incubated for 1 or 24 h. Seven-day-old carrot seedlings, growing on vermiculite beds, were sprayed with a 10-4M solution of ABA and incubated for 1, 4, 8 or 24 h at 25 °C under dim light.
Isolation of RNA from carrot cells In order to construct a cDNA library, total RNA was isolated from clusters of embryogenic cells (14.5 g), which had been cultured in medium supplemented with 2,4-D, by guanidine thiocyanate method [15], and poly(A) + RNA was isolated from the RNA by two cycles of column chromatography on oligo(dT)-cellulose. For northern blot analysis, RNA was isolated from various cells by the phenol/SDS method [2].
Isolation of genomic DNA from carrot seedlings Carrot genomic D N A was extracted from 90 g of three-week-old seedlings by the protocol of Maniatis et al. [ 15].
Preparation of a cDNA library Poly(A) + RNA, prepared from clusters of embryogenic cells, was used to construct a cDNA library in lambda gtl 1 by use of the cDNA Synthesis System Plus and the cloning system from Amersham (Aylesbury, UK). The library was amplified and used for further experiments.
Amplification of target DNA by the polymerase chain reaction (PCR) The amplification of the specific D N A was performed by the polymerase chain reaction (PCR),
241 using the Gene Amp DNA Amplification Reagent Kit (Perkin Elmer Cetus, Norwalk, CT). The primers for PCR consisted of the forward primer, 5 ' -GGIAA(A/G)ACICA(A/G)AA(A/G)GGIG GICC-3', derived from the amino acid sequence GKTQKGGP, and the reverse primer, 5'-GCI ACICCIGG (A/G) TAIGTCATCAT-3 ', derived from the amino acid sequence MMTYPGGVA, which were reported previously [14]. The template for PCR was 100 ng of the DNA from the cDNA library from the clusters of embryogenic cells and either one of the two primers was added at 100 pmol. The denaturing and annealing processes during the reaction were performed at 94 °C for 1 min and 37 °C for 2 min, respectively, and the synthesis of DNA from the primers was catalyzed by the thermoresistant Taq I polymerase at 72 °C for 3 min.
Screening and subcloning of the cDNA library Recombinant phages were plated at high density (50000 plaques per 154 cm 2 dish), transferred onto nylon filters (Dupont, Boston, MA) and screened by hybridization with [32p]-labeled 600 bp PCR fragments using a Random Primed DNA Labeling Kit (Boehringer Mannheim, Mannheim, FRG). Hybridization was performed according to the instructions from the manufacturers of the nylon filters. Lambda DNA was isolated by ultracentrifugation on a CsC12 gradient [15]. The cloned cDNA was isolated from Eco RIdigested lambda DNA, separated on a 0.7 ~o lowmelting-temperature agarose gel and electroeluted from gel slices in an electroeluter (IBI, New Haven, CT). The cDNA was ligated into pBluescript SK- (Stratagene, La Jolla, CA) and M13mpl8 or M13mpl9 phage, and Escherichia coli strains XL-1 and JM109 served as hosts for the plasmid and phage, respectively.
sequenced by the dideoxynucleotide chain-termination method with Sequenase (USB, Cleveland, OH) in accordance with the manufacturer's protocols.
Hybridization of RNA Total RNA (20 ~g) from carrot tissues or cultured cells was denatured with 2.15 M formaldehyde and 50~o (w/v) formamide and then fractionated by electrophoresis in a 1.2~o (w/v) agarose gel that contained 2.2 M formaldehyde [14]. The RNA was subsequently transferred to a nitrocellulose membrane in 20x SSC (1 x SSC -- 0.15 M NaC1, 0.015 M trisodium citrate, pH 7.0) as transfer buffer. The transferred RNAs were hybridized with [32p]-labeled fragments ofcDNA, labeled with the Random Primed DNA Labeling Kit. Prehybridization, hybridization, and posthybridization steps were performed essentially as described elsewhere [15]. The fragments ofcDNA that hybridized to specific RNAs were visualized by autoradiography.
Hybridization of DNA Carrot DNA was digested with restriction endonucleases and the resulting fragments were separated according to size on a 0.7~o (w/v) agarose gel and then transferred to a nitrocellulose membrane for hybridization as described elsewhere [ 15]. The transferred DNAs were hybridized with [32p]-labeled fragments of cDNA, labeled with the Random Primed DNA Labeling Kit and the hybridized fragments were visualized by autoradiography.
In situ hybridization using digoxigenin-labeled
probes DNA sequence analysis The plasmid DNA templates and the singlestranded DNA templates from M13 phage were
Clusters of embryogenic carrot cells and dry fruits (seeds) were fixed with 4 ~o paraformaldehyde in PBS (10 mM sodium phosphate, 150 mM sodium chloride, pH 7.0) for 12 h at 4 °C. The fixed sam-
242 ples were embedded in O.C.T. compound (Miles Scientific, Naperville, IL) and frozen at -80 ° C. Sections (20 #m) were cut at -25 °C on a cryostat and transferred to glass slides, which had been coated with a 2~o solution of Vectabond reagent (Vector Laboratories, Burlingame, CA) in acetone. Prior to synthesis of RNA, the plasmids containing ECP31 cDNA were linearized with Xho I (when T3 polymerase was used) or Xha I (when T7 polymerase was used). Antisense and sense RNA probes were transcribed from T3 and T7 promoters of the recombinant pBluescript using a digoxigenin-labeling kit (Genius Kit; Boehringer Mannheim). The prehybridization treatments, the hybridization with RNA probes, the posthybridization treatments and the immunological detection were performed essentially as described in the Technical Bulletin from Boehringer Mannheim Biochemicals. After the color had developed, the sections were rinsed for 30 min with two changes of distilled water and mounted with 50~o glycerol that contained 7~o gelatin. Photographs were taken under a light microscopy, Vanox model AHBT (Olympus, Tokyo, Japan).
from the proteolytic fragments of ECP31 on a gas-phase protein sequencer [14]. The amplified 600 bp fragments of D N A were used as a probe for screening the cDNA library. A total of 300 000 phages were screened and 12 cDNA clones were isolated. Eight clones had inserts of about 1.05 kb, two had inserts of 0.8 kb and two contained a fragment of 2.7 kb. Partial sequencing from both the 5' and the 3' termini showed that there were at least two rather similar kinds of ECP31 cDNA of 1.05 kb and that the 0.8 kb fragments were truncated forms of the 1.05 kb fragments. The 2.7 kb fragments were found to be artifacts derived from combinations of ECP31 cDNAs and ribosomal DNA. A 1.05 kb fragment was subcloned into the Eco RI site of pBluescript and the resultant plasmid was designated pcECP31.
Gel-blot analysis of genomic DNA with pECP31 Genomic D N A from three-week-old carrot seedlings was digested with Hind III, Bam HI and Eco RI. Hybridization was carried out at 42 °C with [32p]-labeled pcECP31 (Fig. 1). After digestion with Hind III and with Barn HI, 3.9 kbp and
Results
Amplification by PCR of target DNA and isolation of cDNA that encoded ECP31 Poly(A) + RNA was isolated from clusters of embryogenic carrot cells and used for construction of a cDNA library with the lambda gtl 1 vector. In order to obtain a probe for screening of the c D N A library and isolation ofcDNAs for ECP31, polymerase chain reactions (PCRs) were carried out using 100 ng of the isolated DNAs from the c D N A library at an annealing temperature of 37 ° C, for 30 cycles, with the primers GGIAA(A/ G) ACICA (A/G) AA (A/G) G G I G G I C C and GCIACICCIGG (A/G) TAIGTCATCAT, derived from the amino acid sequences GKT Q K G G P and MMTYPGGVA, respectively. These sequences had been previously determined
Fig. 1. Genomic Southern blot analysis ofECP31 genes. Total DNA was digested with the restriction endonucleases indicated and probed with the [32P]-labeled cDNA of pcECP31. H, Hind III; B, Barn HI; E, Eco RI. Fragments of Hind IIIdigested lambda DNA were used as size markers.
243 4.2 kbp bands were detected, respectively, and two hybridizing bands of 6.5 kb and 15 kb were detected after digestion with Eco RI. Since at least two different but similar cDNA clones were obtained by the screening, it is likely that there are two or three genes for ECP31 within about 4 kb of carrot genomic DNA.
Accumulation of ECP31 mRNA during somatic embryogenesis
Northern blot analysis was used to determine the levels of ECP31 m R N A during somatic embryogenesis. The results are shown in Fig. 2. Clusters of embryogenic cells were found to contain a high level of ECP31 mRNA. When somatic embryogenesis was induced by transfer of the embryogenic cells to medium without 2,4-D, the level of this m R N A decreased and little of this m R N A was present at detectable levels in torpedo-shaped somatic embryos. In situ hybridization demon-
Fig. 2. Northern blot analysis of ECP31 mRNA during formation of somatic embryos. Total RNA was extracted from embryogenic cells (lane 1), somatic embryos at the heart stage, which correspond to those on the 10th day of culture after transfer of embryogenic cells to auxin-free medium (lane 2) and somatic embryos at the torpedo stage, which correspond to those on the 14th day of culture after transfer (lane 3). Twenty #g each of total RNA were separated on a 1.2% agarose gel that contained 2.2 M formaldehyde. The RNAs were subsequently blotted onto a nitrocellulose membrane and probed with [32P]-labeled cDNA from pcECP31.
strated that ECP31 transcripts were preferentially localized in the peripheral cells of the clusters of embryogenic cells (Fig. 3), where pro-embryos originated [12]. Because cell density of the outer part of the cluster is not significantly different from that of the inner part, the peripheral embryogenic cells accumulated more ECP31 transcripts than other cells of the cluster.
Analysis of the nucleotide and amino acid sequences
The 1012 bp nucleotide sequence of pcECP31 is shown, together with the deduced amino acid sequence, in Fig. 4. After the stop codon, pcECP31 has a 197 bp 3'-non-coding region which is followed by 19 adenyl residues and the sequence AATAAG which resembles the AATAAA motif 75bp upstream from the polyadenyl tail. pcECP31 contains an open reading frame of 256
Fig. 3. Localization of ECP31 mRNA in clusters of embryogenic cells by in situ hybridization. Sections of clusters of embryogenic cells were hybridized with single-stranded digoxigenin-labeled sense (A) and antisense (B) RNA probes of ECP31 cDNA.
244 i0 20 30 40 50 60 70 80 90 C~&~AG~a~ATTTACACACAAATCCTC`&gC~&~a~AATACAAAGAT~A~CCA~C~C~CCAC~AGACCC~C~G~A~CCCATCAAATAC~A M S Q Q Q P R R P Q Q E Q P I K Y G
i00 D
ii0 120 130 140 150 160 170 180 190 c~TATTT~AT~TTTCC~cA~TT~AGCTCCC~CCC~TCGCCCCc~C~AC~CCTcc~CcAT~CA~cTGcG~AG~TAT~T~CTTG~T~%GACGCAG V F D 47 S G Q L S S Q P V A P N D A S A M Q A A E N M V L G 210 220 230 240 250 260 270 280 ~GGT~TCC~CCTCTGTcAT~CA~Tc~C~c~CT~CT~TCTGCA~CGC~C~Tc~TTG~CCcGCAcGA~cACCCCcATA~CTA~T~C~ K G G P A S V M Q S A A A A N L Q R G V V G P H E G T P I A S
200 K
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400
410 420 430 440 450 460 470 480 490 ~ATGCC~CCC~cTGGT~T~cTCG~cA~T~ATTCGATTACTATAGGC~AG~CTTT~A~ACCACTGCCTT~Cc~cCG~AGAT~GCCTGTA~ATCAG M P S P A G V L G S D S I T I G E A L E T T A L T A G D K P V D Q
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510 520 530 540 550 560 570 580 590 A~T~AT~CA~TGCTATAC~CA~TG.~T~AGA~C~T~CTAT~TTATCCT~T~A~TG~C~CAG~T~CTC~TCT~C~C~ATTAT~T~
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610 620 630 640 650 660 670 680 cTC~TACCAT~T~TT~CCA~C~CT~GCTC~TGATGTcCTT~CT~ATC~ATc~ATTAG~TT~CT~AC~C~T~AC~AGGG~AT~C R T M N V A S K T K L G D V L A D R S I R L A E D K A 710
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810 820 830 840 850 860 870 880 890 ~ACCC~CTTTCT~TATTCCAT~T`~%AcATATCTATc~GTATTATTATGTAG~T~TGT~TTTTCAT~TACA~T~TTT~A~`~T~TA~TATcTATT D P T F ~ 910
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~TCATATTGTGTGTAT@--~GCTTGCGTACTA~TTGATG~AGTGCGTGTG~ACTGCTGTTTTGGTTG~TGC~GGG~TGGTTTTGGTGT~ I010 AAAAAAAAAAAA
Fig. 4. Nucleotide sequence of the cDNA ~ r ECP31 and the deduced amino acid sequence. The amino acid sequences identical to those ~ u n d in purified ECP31 are underlined. The putative polyadenylation s i s a l is boxed.
amino acids which begins with a methionine codon, and the calculated relative mass of this polypeptide is 26 111. The five sequences of amino acids that were previously determined by proteolytic digestion on membrane and subsequent analysis on a protein sequencer [14] are shown by underlines. The polypeptide has compositional bias towards Ala (16.86~) and Gly (10.20~) and it lacks Cys and Trp residues. The predicted amino acid sequence encoded by cECP31 was used in a search for homologous proteins in the SWIS S-PROT (European Molecular Biology Laboratory, Heidelberg, FRG) protein database, release 14, using the similarity search program. As a result, extensive homology (62.2~o) was found between ECP31 and lateembryogenesis-abundant (LEA) protein D34 of cotton [3] (Fig. 5).
Expression of the gene for ECP31 in plants Since the expression of the gene for LEA D34 was detected at the late stages of the formation of cotton seeds [3, 10], we were prompted to examine the expression of the gene for ECP31 during the maturation and germination of carrot seeds. ECP31 transcripts were not detected from 3 days to 23 days after anthesis but they began to be found from 28 days after anthesis (Fig. 6A). Relatively large amounts of ECP31 m R N A accumulated in dry fruits (seeds) but this m R N A became undetectable within 24 h after imbibition (Fig. 6B). The difference of the size of the hybridized mRNAs between seeds and embryogenic cells may be due to the difference of the modification of the m R N A or that embryogenic cells have electrophoresis-disturbing substances. In
245 ECP31
i' MSQQQPRRPQQ . . . . . . .
LEA D34
1" MSQGQPRRPQQPAGQGENQEPIKYGDVFNVSGELANKPIAPQDAAMMQTAETQVLGQTQK
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232'
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Fig 5.
C o m p a r i s o n of amino acid sequences between ECP31 and L E A D34. C o m m o n amino acids are indicated by asterisks and similar amino acids are i n d i c t e d by dots.
situ hybridization using a digoxigenin-labeled RNA probe revealed the embryo-specific accumulation of ECP31 m R N A in seeds (Fig. 7).
Regulation by ABA of the accumulation of ECP31 mRNA ABA may play a role in the regulation of expression of some classes of genes at late embryonic stages [11, 21]. To investigate the effect of ABA on the accumulation of ECP31 mRNA, RNA was extracted from torpedo-shaped somatic embryos which had been grown for 13 days without 2,4-D. Prior to the extraction, ABA (3.7 x 10-6 M) was added to the culture medium and somatic embryos were collected at 1 and 24 h after the start of this treatment. As shown in Fig. 8, little level of ECP31 transcripts was found in control somatic embryos, but the level increased during the 24 h that followed the application of ABA up to the same level in embryogenic cell clusters. Next we investigated whether or not the effect of ABA on expression of the ECP31 gene was embryo-specific. Seven-day-old carrot seedlings were sprayed with a 10-4M solution of ABA and collected at 1, 4, 8, or 24 h after this treatment. Northern blot analyses demonstrated that there was no detectable ECP31- specific m R N A in any of the ABA-treated carrot seedlings (data not shown).
Discussion
ECP31 is an intracellular protein isolated from clusters of embryogenic carrot cells, with a relative mass of 31000. It accumulates in clusters of embryogenic cells and disappears in the course of formation of somatic embryos. The appearance of ECP31 in hypocotyl segments cultured on MS semisolid medium supplemented with 2,4-D coincides with the formation of embryogenic callus. ECP31 in embryogenic callus was demonstrated to be located preferentially in the peripheral regions, in which pro-embryos are known to originate [ 12]. Given these results, we postulated previously that ECP31 performs important role(s) in the induction and/or maintenance of embryogenic competence [13]. In this report, we describe the isolation of a c D N A that encodes ECP31 and an examination of expression of the gene for ECP31 using the cDNA as a probe. First, we performed PCR using oligonucleotide primers derived from two previously determined internal sequences of amino acids in ECP31, and an amplified 600 bp fragment was used as a probe for screening the c D N A library from cultured embryogenic cells. Among 300000 recombinant phages screened, 12 clones were isolated and finally the cDNA from one clone (full-length clone) was used for further experiments. Results of genomic Southern hybridization, as well as sequence analysis of cDNA clones, dem-
246
Fig. 6. Northern blot analysis of ECP31 mRNA during development (A, upper) and germination (B, lower) of seeds. Total RNA was extracted from fruits (seeds) or seedlings of carrot. Twenty/~g of each RNA were separated on an agarose gel, blotted onto a membrane and probed with [32P]-labeled cDNA of pcECP31. A. Lane 1, fruits 3 days after anthesis (3DAF); Lane 2, 8 days; lane 3, 13 days; lane 4, 18 days; lane 5, 23 days; lane 6, 28 days after anthesis. B. Lane 1, dry fruits (seeds); lane 2, seeds 1 day after sowing; lane 3, 2 days; lane 4, 3 days; lane 5, 4 days; lane 6, 5 days; lane 7, 7 days. Twenty #g of RNA from clusters of embryogenic (EG) cells were indicated as a positive control (lane 8).
onstrated that D. carota has at least two related genes that encode ECP31. This result supports the result that ECP31 can be resolved into two polypeptides, each with a relative mass of 31000, by two-dimensional PAGE (unpublished results). A large amount of ECP31-specific mRNA was found in embryogenic cell cultures but the level of this m R N A decreased and finally it became undetectable at the torpedo stage, during the course of the development of somatic embryos. In situ
hybridization demonstrated that transcripts of the gene for ECP31 were preferentially situated in the peripheral region s of cluster s of embryogenic cells. These results were essentially the same as those obtained with an ECP31-specific antibody [13]. Thus, not only the accumulation of ECP31 but also the expression of its gone is correlated with the embryogenic competence of carrot cells. The DNA complementary to ECP31-specific mRNA consisted of 1015 bp and the amino acid sequence of the protein was deduced to be that of a polypeptide with a Mr of 26 111. The reason for the difference in size from that obtained by SDSPAGE (31000) may be related to some modifications, such as kinations (ECP31 has six putative casein kinase II phosphorylation sites and two putative protein kinase C phosphorylation sites), or to the nature of the protein. The search of the database revealed that ECP31 exhibits significant homology at the nucleotide level (42.5 ~o) and at the protein level (62.2~o) to the LEA D34 clone [3]. LEA mRNAs, which were isolated initially from cotton cotyledons [3, 10], are expressed of high levels at late embryonic stages and their levels decrease during the course of germination of seeds. Recent studies demonstrated that not only cotton but also other plant species, such as carrot, barley, rice, rape and wheat, have LEA mRNAs [9, 21]. From the conservation of various sequences, these clones have been classified into three groups: group 1, wheat Em and cotton D19; group 2, rice rabl6 (formerly rab21), cotton D l l and barley dehydrin; group 3, cotton D7, carrot Dc3, carrot Dc8, barley pHVal and rape pLEA 76 [9]. Our present results indicate the existence of an additional fourth group of Lea clones, namely cotton D34 and carrot pcECP31, in the plant kingdom. Some LEA proteins have been predicted to protect plant cells from water stress, on the basis of the patterns of their expression and their extensive hydrophilic characteristics [9]. However, ECP31 and D34 do not have such hydrophilic characteristics. This difference implies that they are probably functionally different from the other LEA proteins. The expression of the genes for cotton LEAs,
247
hybridization of the digoxigenin-labeledECP31 RNA probe in carrot seeds. Section from dry carrot fruits (seeds) were hybridized with single-stranded digoxigenin-labeled sense (A) and antisense (B) RNA probes of ECP31 cDNA. EM, embryo; EN, endosperm; OD, oil duct; P, pericarp. Bar = 100 #m. Dark purple signal found in the zygotic embryo indicates the existence of ECP31-mRNAs. Black color of the cell walls of endosperm and pericarp is due to the focus.
Fig. 7. In situ
including D34, was reported to be highly induced by treatment with ABA in premature zygotic embryos [ 10], but their organ specificity has not yet been reported; their homologues, such as rabl6, barley dehydrin and wheat Era, are induced by ABA without organ specificity [7, 16, 17, 21, 27]. The northern analysis of m R N A for ECP31 showed that the m R N A accumulated at late stages of embryogenesis, as in the case of cotton LEAs and their homologues, and the m R N A was located only in zygotic embryos (Fig. 8). Moreover, as shown in Fig. 9, treatment with ABA highly enhanced expres sion of the gene for ECP31 in torpedo-shaped somatic embryos, as reported
in cotton zygotic immature embryos, but not in carrot seedlings. This embryo-specific expression is unique among LEA m R N A s examined to date, and our present results suggest that both embryospecific factor(s) and ABA are involved in the expression of the gene for ECP31. The high level of expression of the gene for ECP31 in embryogenic cells implies the existence of both embryospecific factor(s) and of a high level of endogenous ABA in those cells. In preliminary experiments, endogenous level of ABA in embryogenic cells was estimated to be higher than that in somatic embryos, and was comparable to that in mature carrot seeds. Thus, the characteristic of
248
References
Fig. 8. Effect of ABA on expression of the gene for ECP31 in carrot somatic embryos. Total RNA was extracted from somatic embryos, fractionated on an agarose gel, blotted onto a membrane and probed with [32p]-labeled cDNA ofpcECP31. Twenty/~g of total RNA were loaded in each lane. Lane 1, control, 15 days after the transfer to medium without 2,4-D. ABA was added to the medium and samples were collected after 1 h (lane 2) and 24 h (lane 3).
E C P 3 1 a n d o t h e r c a r r o t e m b r y o n i c genes, s u c h as D c 3 a n d D c 8 , b e i n g e x p r e s s e d a l r e a d y at t h e e m b r y o g e n i c cell c l u s t e r stage, while t h e y o n l y a p p e a r m u c h l a t e r in z y g o t i c d e v e l o p m e n t , m a y b e d u e to t h e high level o f e n d o g e n o u s A B A in t h e e m b r y o g e n i c cell clusters.
Acknowledgements T h i s r e s e a r c h w a s s u p p o r t e d in p a r t b y G r a n t s i n - A i d for S p e c i a l R e s e a r c h o n P r i o r i t y A r e a s ( P r o j e c t 02242102, C e l l u l a r a n d M o l e c u l a r B a s i s for R e p r o d u c t i v e P r o c e s s e s in P l a n t s ) f r o m t h e Ministry of Education, Science and Culture, Japan, and by Special Coordination Funds of the Science and Technology Agency of the Japanese Government.
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