Plant Cell Reports (2000) 19 : 739±747

 Springer-Verlag 2000

C E L L B I O L O G Y A N D M O R P HO G E N E S IS

S.-K. Tan ´ H. Kamada

Initial identification of a phosphoprotein that appears to be involved in the induction of somatic embryogenesis in carrot

Received: 1 October 1999 ´ Accepted: 3 November 1999

Abstract We examined changes in the absence versus presence patterns of phosphoproteins with respect to the acquisition of embryogenic competence during somatic embryogenensis in carrot (Daucus carota L.). To characterize a possible correlation between the induction of embryogenic competence and protein phosphorylation, we examined the patterns of protein phosphorylation in embryogenic cells (EC) and nonembryogenic cells (NC) that had lost the ability to form somatic embryos. Two-dimensional polyacrylamide gel electrophoresis and subsequent autoradiography revealed the presence of 31 phosphoproteins in EC but not in NC. Furthermore, when we examined the induction of somatic embryogenesis by certain stress compounds in the absence of phytohormones, we identified one specific phosphoprotein (ECPP-44). ECPP-44 was found to be induced in all treatments that resulted in embryogenic competence. The partial amino acid and nucleotide sequence of ECPP-44 shows partial homology to two dehydrins (ERD10 and ERD14) from Arabidopsis. Key words Daucus carota ´ Embryogenic competence ´ Protein phosphorylation Abbreviations ABA: Abscisic acid ´ CBB: Coomassie brilliant blue ´ DTT: Dithiothreitol ´ EC: Embryogenic cells ´ LEA: Late embryogenesis abundant ´ MS: Murashige and Skoog©s medium ´ NC: Non-embryoCommunicated by T. Yoshikawa S.-K. Tan ()) ´ H. Kamada Gene Experiment Center, Institute of Biological Sciences, University of Tsukuba, Tennoudai 1 ± 1-1, Tsukuba, Ibaraki 305 ± 8572, Japan Fax: 81 ± 298 ± 536006 e-mail: [email protected] Supplementary material Figures 6 and 7 has been deposit in electronic form and can be obtained from http://link.springer.de/link/service/journals/299

genic cells ´ PVDF: Polyvinylidene difluoride ´ SDS: Sodium dodecyl sulfate ´ 2,4-D: 2,4-Dichlorophenoxyacetic acid ´ TCA: Trichloroacetic acid ´ Tris: TRIS-hydroxymethylaminomethane ´ RT-PCR: Reverse transcription polymerase chain reaction

Introduction Somatic embryogenesis in carrot (Daucus carota L.) can be divided into two main process, namely, the process whereby differentiated somatic cells undergo certain physiological changes to acquire embryogenic competence and proliferate as embryogenic cells in auxin-containing medium and the process whereby the embryogenic cells display their embryogenic competence and differentiate into somatic embryos. Phosphorylation, a common and important mechanism in the reversible regulation of specific proteinsmay be involved in the acquisition of embryogenic competence. In plants, the responses of cells or tissues to external stimuli, such as light (Raymond and Douglas 1990), phytohormones (Raz and Fluhr 1993; Mizoguchi et al. 1994) and environmental stress (Suzuki and Shinshi 1995; Reddy and Prasad 1995; Kyo and Harda 1990; Yupsanis et al. 1994), are mediated in part by the expression of genes whose products contribute to a given physiological effect. Various genes involved in protein phosphorylation during the development of living cells have been isolated and characterized (Lynn and Walker-Simmons 1995; Mizoguchi et al. 1993, 1994; Stafstrom et al. 1993; Wilson et al. 1993; Kieber et al. 1993). However, our knowledge of the regulatory network governing growth and developmental responses is fragmentary, with almost no information available on the interaction with the phosphorylation status of proteins. During the course of our ongoing investigation of protein control during embryogenesis in carrot, we have reported that the formation of embryogenic cells from carrot somatic cells can be triggered by a variety

740

of procedures and exposure to various stress compounds (Kiyosue et al. 1989, 1990; Tachikawa et al. 1998). Proteins specific to embryogenic cells have been isolated using such systems (Tachikawa et al. 1998). However, the molecular mechanisms by which somatic cells acquire embryogenic competence have not yet been clarified. Here we report the phosphorylation of a protein, named ECPP-44, that was present in all the treatments with all the stress compounds tested, and from which embryogenic cells were acquired. Based on the partial amino acid sequence of ECPP-44, degenerate primers were designed to PCRamplify a fragment of the ECPP-44 cDNA. The cDNA contains a lysine-rich element and eight serine residues, and shares extensive homology with other dehydrins. The presence of a positive relationship between the phosphorylation of ECPP-44 and the acquisition of embryogenic competence is discussed.

Labeling in vivo with [32P]-orthophosphate Both EC and NC were radiolabeled by incubation with [32P]-orthophosphate 2 days after subculture. In order to promote the uptake of [32P]-orthophosphate, cells (2.0 mg fresh weight) were cultured for 3 h in a 1.5-ml Eppendorf tube that contained 1.0 ml fresh phosphate-free MS basal medium plus 2,4-D (1 mg/l) from which KH2PO4 had been omitted. Cells were labeled by incubation for 3 h in MS basal medium that contained 2,4-D (1 mg/l) and carrier-free [32P]-orthophosphate (9.25 MBq/ml; ICN, USA). Somatic embryos were labeled in the same way, with the exception that phosphate-free MS basal medium without 2,4-D was used instead of phosphate-free medium with 2,4-D. Shoot apices that had been treated with various compounds (a high concentration of sucrose, NaCl, CdCl2, 2,4-D or ABA) were incubated for 4 h in fresh phosphate-free MS basal medium with the indicated compound and then for 3 h in MS basal medium with the same compound and carrier-free [32P]-orthophosphate (14.23 MBq/ml). Labeling was terminated by removing the radioactive medium and then rinsing the cells or shoot apices three times with fresh phosphate-free basal MS medium. Preparation of samples

Materials and methods Plant materials, cell culture and induction of somatic embryo formation Seeds of Daucus carota L. cv. US-Harumakigosun were allowed to germinate in moist vermiculite at 25 C under 16 h of illumination supplied by white fluorescent lights (approximately 40 mmol photons/m ± 2 s ± 1) daily. One-week-old seedlings were surface-sterilized with a 1% sodium hypochlorite solution, and its hypocotyls (approx. 1 cm in length) were subsequently cut off and cultured in plastic petri dishes (9 cm in diameter) containing 30 ml of semi-solidified (0.8%, Agar powder, Wako) MS basal medium, pH 5.7 (Murashige and Skoog 1962), to which 1 mg/l 2,4-D was added for 1 month. The yellowish nodular callus that formed on each hypocotyl explant was suspended at 2-week intervals in 300-ml flasks containing 100 ml of liquid MS basal medium with 1 mg/l 2,4-D. To obtain somatic embryos, we transferred the small cell clusters of EC (37±63 mm in diameter) into 2,4-D-free MS basal medium. Embryos that had formed after 2 weeks of culture were filtered through a stainless-steel mesh (pore size, 500 mm), which enabled the collection of torpedo-stage embryos on the mesh (Satoh et al. 1986). Cell clusters less than 1 mm in diameter were filtered from 1-week-old EC suspensions and subcultured for 2 weeks in liquid MS basal medium with 1 mg/l 2,4-D. This procedure was repeated for 2 years, and the resultant cells, which had lost the ability to form somatic embryos, were designated non-embryogenic cells (Satoh et al. 1986) and used as a negative control. Induction of somatic embryogenesis by stress compounds Seedlings of carrot were prepared and surface-sterilized with a 1% solution of sodium hypochlorite under aseptic conditions as described above. Shoot apices (approx. 1 cm in length) cut from 10- to 13-day-old carrot seedlings were cultured for 2 or 4 weeks on semi-solidified (0.8%) agar MS basal medium supplemented with stress compounds to which no phytohormones were added. They were subsequently transferred onto phytohormone-free MS basal medium without stress compounds. The effective chemicals and duration of treatment for somatic embryogenesis induction were as follows: 0.7 M sucrose for 4 weeks (Kamada et al. 1993); 0.3 M NaCl for 4 weeks (Kiyosue et al. 1989); 0.6 mM CdCl2 for 4 weeks (Kiyosue et al. 1990); 10 ± 4 M ABA for 2 weeks (Kamada and Harada 1981); and 1 mg/l 2,4-D for 4 weeks.

Labeled cells (2.0 mg), explants (150 of shoot apices) or somatic embryos (2.0 mg) were pulverized with a glass pestle in an Eppendorf tube that contained 0.2 ml of ice-cold extraction buffer [10 mM TRIS-HCl (pH 7.0), 10 mM MgCl2, 50 mM KF, 1% (v/v) Nonidet NP-40, 1 mM DTT, 50 mg/ml RNase A, 100 mg/ml DNase I, 1 mM phenylmethylsulfonyl fluoride, 1 mM NaVO3, 0.1% (w/v) SDS]. A 0.2-ml aliquot of the homogenate was transferred to a 2.0-ml Eppendorf tube and 1.8 ml of a 10% TCA solution that contained 100% acetone mixture solution was added. The mixture was allowed to stand on ice for 30 min, and precipitated proteins were pelleted by centrifugation at 12,000 g for 15 min at 4 C. The pellet was washed three times with ice-cold 100% acetone. The washed pellet was vacuumdried, and the proteins were resuspended in lysis buffer [5 M urea, 1 M thiourea, 0.1 M DTT, 2% (v/v) Nonidet NP-40, 5% (v/v) glycerol, 2% (v/v) ampholytes (1.6% pH 5±7 and 0.4% pH 3.5±10; Phamarcia Biotech AB, Sweden)]. The radioactivity in the suspension was measured in a liquid scintillation counter by the TCA-insoluble assay method (Ono et al. 1991). Total protein was quantified by the method of Bradford (1976) using the Bio-Rad dye-binding assay (Bio-Rad, USA) with BSA (Sigma, USA) as the standard. Two-dimensional gel electrophoresis The procedures for two-dimensional gel electrophoresis were a modified version of that described by O©Farrell (1975). [32P]-labeled proteins (aliquots containing 300,000 cpm) were loaded on the isoelectric focusing (IEF) gel (13 cm in length; 5.5% acrylamide/0.3% methylenebisacrylamide) for electrophoresis in the first direction. The anode (upper) and cathode (lower) buffers were 20 mM NaOH and 10 mM H3PO4, respectively. Electrophoresis was performed for 12 h at 400 V and then for 1 h at 800 V. After electrophoresis, the gel was allowed to equilibrate for 20 min in 62.5 mM TRIS-HCl (pH 6.8) that contained 2.0% SDS, 10% glycerol, 50 mM DTT and 3 mM bromophenol blue. Polyacrylamide gel electrophoresis for fractionation in the second dimension was performed as described by O©Farrell (1975) with an 11% acrylamide running gel. After electrophoresis for 4.5 h at 25 mA, the proteins on the gel were stained with a silver-staining kit (Wako Chemical Co, Japan). The gel was then dried and exposed for autoradiography at ± 80 C to X-ray film (Kodak, Japan) or to the imaging plate of a Bioimage Analyzer (BAS2000; FUJIX, Japan) with an intensifying screen. The duration of exposure was varied to allow for the detection of major and/or minor spots. The 2-D gel assay was repeated at least tree times per treatment.

741 Peptide mapping of a 44-kDa phosphoprotein The band of a [32P]-labeled 44-kDa phosphoprotein (ECPP-44) was excised from a gel after two-dimensional electrophoresis. The excised fragment of gel was then loaded with V8 proteinase (Sigma, USA) into the well of an SDS-polyacrylamide (18% polyacrylamide) gel. After 30 min, which allowed for partial digestion of the protein, electrophoresis was performed for 4 h at 25 mA. The peptides on the gel were first stained with the silver-staining kit, and the gel was then dried and exposed for autoradiography to the imaging plate of the Bioimage Analyzer with an intensifying screen. For amino acid sequence analysis, instead of silver staining, the gel was equilibrated with an electroblot running buffer [48 mM TRIS, 39 mM glycine and 20% methanol] (Bjerrum and Schafer-Nielsen 1986), and proteins were blotted onto a PVDF membrane (Immobilon-P; Millipore, USA) using a semi-dry transfer cell (Bio-Rad). Electroblotting was carried out at a constant current of 200 mA for 70 min. Four peptide bands (ec-1, ec-2, ec-3 and ec-4) were visualized after staining with CBB and subsequently excised from the PVDF membrane for amino acid sequencing (Procise 492 automatic sequencer; Applied Biosystems, USA). A search for protein homology to the sequences of the four internal peptides was made using the DDBJ and SWISS-PROT databases. 39-RACE (RT-PCR) Two micrograms of total RNA from EC was amplified at 42 C for 30 min, 99 C for 5 min and 5 C for 5 min with StrataScript RT-PCR reaction buffer containing StrataScript buffer [50 mM TRIS-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM DTT], 25 mM dNTP mixture, 80 U RNase Block I, 50 U StrataScript RNase H-reverse transcriptase, 0.2 mg random primer (36 mer) and RNase-free H2O. PCR amplification of embryogenic cell cDNA was carried out with two degenerate primers which were designed essentially by partial amino acid sequences (ec-1 and ec-3) of ECPP-44. ECPP-S (59-GAYTGYAARGTIGTIGARGARGARG-39) and ECPP-A (59-TTYTTYTTYTTYTTYTCICCICC-39) were designed as forward and reverse primers, respectively. RoboCycler (Stratagene) gradient PCR was carried out with 35 cycles: 1 min at 94 C, gradient temperature ranges (53±63 C) for 2 min and extension at 72 C for 3 min, followed by a final extension for 15 min at 72 C. The expected band which appeared at 53 C was cut out from the gel, and the DNA was extracted from the gel slices by the crush and soak method. Using the same PCR cycling parameters as described above except for 25 cycles and annealing at 53 C, we purified and subcloned the reamplified PCR product into the TA-cloning vector (pECPP-S+A). A DNA Sequencer Model 373A (ABI, San Jose, Calif.) was used for DNA sequencing. Nucleotide and amino acid sequences were analyzed using the GENETYX software system (Software Development Co, Tokyo, Japan).

Results Labeling of proteins in vivo and analysis of the phosphoproteins in embryogenic cells and non-embryogenic cells One hundred and sixty-five phosphoproteins were detected when EC were labeled in vivo in MS basal medium supplemented with 2,4-D (Fig. 1A, B) and 157 phosphoproteins were detected in NC (Fig 1C, D). Three phosphoproteins were consistently found in EC, NC and stress- or no-stress-treated shoot apices (arrows in Figs. 1 and 2). Consequently, these phos-

phoproteins were used as positional markers. Close inspection of the autoradiograms revealed that 31 phosphoproteins were present in EC but absent from NC (Fig. 1). To determine whether the phosphoproteins found specifically in EC might be related to the acquisition of embryogenic competence, we examined the absence versus presence patterns of phosphoproteins in detail after the induction of somatic embryogenesis by various stress treatments. Phosphoproteins following the induction of somatic embryogenesis by various stress treatments Among the 31 phosphoproteins detected specifically in EC but not in NC, 15, 13, 13, 20, 13 and 15 phosphoproteins were found following the treatment of shoot apices with sucrose, NaCl, CdCl2, 2,4-D, ABA and no stress (shoot apices were cultured for two weeks without any stress treatment; Fig. 2, Table 1). In particular, a phosphoprotein (no. 10) of about 44 kDa (hereafter referred to as ECPP-44) was found in all stress-induction treatments but not in control cultures or in NC (Table 1). These observations suggested that ECPP-44 might be related to the acquisition of embryogenic competence. Phosphoproteins in developing somatic embryos When proteins in developing somatic embryos were radiolabeled in vivo with [32P]-orthophosphate, we detected only 24 of the 31 phosphoproteins originally found to be specific to EC. Thus, 7 phosphoproteins (nos. 8, 9, 12, 13, 14, 15 and 16) were absent during the later stage of embryo development (i.e. they were not detected in torpedo-staged embryos). However, ECPP-44 was detected in torpedo-staged embryos (data not shown). Detection of ECPP-44 on gels after silver-staining Close inspection of silver-stained gels from three replicated experiments revealed that ECPP-44 was present in EC (Fig. 3A), of shoot apices that had been treated with various stress compounds and in control cultures (shoot apices that had been cultured for 2 weeks without stress treatment; Fig. 3C), but not in NC (Fig. 3B). ECPP-44 was detectable as a faint silver-stained spot in the analysis of control cultures (Fig. 3C) although it was present at low levels. No autoradiographic signal was detected on the same gel at that position (Fig. 3E, F).

742

Fig. 1A±D Detection of phosphoproteins from embryogenic cells (A,B) and non-embryogenic cells (C,D) on two-dimensional gels. Arrows indicate phosphoproteins consistently detected in all of the cells examined, irrespective of embryogenic competence. The circles with numbers indicate phosphoproteins that were detected in EC but not in NC. A and C show the autoradiograms at a reduced insensity, while B and D show the original autoradiograms from samples that contained 300,000 cpm each

Partial amino acid sequence of ECPP-44 When intact ECPP-44 that had been electroblotted onto a PDVF membrane was subjected to amino acid sequencing, no clear signals were detected, probably as a result of amino-terminal blockage of ECPP-44. Therefore, we determined several internal sequences of ECPP-44 after in-gel digestion of the protein with V8 proteinase. Four peptide fragments (ec-1, ec-2, ec-3 and ec-4) were detected by staining with CBB, and autoradiography revealed that two of them (ec-1 and ec-2) were phosphorylated (Fig. 4). The bands of the four peptides were cut out of the PVDF membrane, and the peptides were sequenced with an auto-

matic sequencer. The amino acid sequence of ec-2 (KAAKPSLLEKL) was identical to that of half of ec-1 (DCKVVEEEEEKAAKPSLLEKL). Thus, ec-2 was probably derived from ec-1. Together, ec-3 and ec-4 (VAPPPPPAAAPVDCAVEGD) formed only one band, as visualized by staining with CBB, but two different sequences were obtained. The ec-3 fragment contained a lysine-rich sequence (GGEKKKKKKEKKGLKEKIEEK) that is characteristic of dehydrinlike proteins. The sequences of ec-1 (66.7%; 47.1%), ec-3 (75.0%; 57.2%) and ec-4 (26.7%; 53.9%) exhibited partial similarity to parts of the predicted amino acid sequences of two dehydrins, group II LEA proteins from Arabidopsis thaliana (ERD10 and ERD14; Kiyosue et al. 1994; Fig. 5). Sequence analysis of the ECPP-44 cDNA fragment When the nucleotide sequences of ec-1, ec-2 and ec-3 were compared with the dehydration and cold stressinducible gene sequences of Arabidopsis thaliana (Kiyosue et al. 1994), we found ec-1 and ec-3 to be quite closely related, which allowed us to use PCR to isolate

743

Fig. 2A±F Patterns of the two-dimensional fractionation of phosphoproteins after the induction of somatic embryogenesis by various stress treatments. Shoot apices were cultured on MS basal medium that contained 0.3 M NaCl for 4 weeks (A,B); 0.7 M sucrose for 4 weeks (C,D), 0.6 mM CdCl2 for 4 weeks (E,F), 1 mg/l 2,4-D for 4 weeks (G,H), 10 ± 4 M ABA for 2 weeks (I,J), and on MS basal medium without any stress compounds (control culture) for 2 weeks (K,L), respectively. Arrows indicate phosphoproteins that were detected consistently in all of the explants/tissues examined. Circles with numbers correspond to the phosphoproteins indicated in Fig. 1. A, C, E, G, I and K show autoradiograms at a reduced intensity; B, D, F, H, J and L show the original autoradiograms obtained from samples that contained 300,000 cpm each. Squares (m) without numbers indicate the absence of phosphoprotein no. 10 in the control culture (K,L)

fragment sequences from carrot. Following PCR amplification, we obtained a 141-bp fragment. The 141-bp nucleotide sequence of ECPP-44 cDNA (DDBJ accession number AB010898) is shown together with the deduced amino acid sequences in Fig. 5. The PCR product contained a cluster of serine residues, synonymous with the amino aicd sequences of group II LEA proteins in Arabidopsis thaliana.

744 Table 1 Summary of the presence versus absence of 31 phosphoproteins found in embryogenic cells (EC) but not in not-embryogenic cells in various cells and stress-treated shoot apices (shoot) Cell type or treatment

Phosphoprotein (spot no.)c 1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

ECa (+1 mg/l 2.4-D)

p

p

p

p

p

p

p

p

p

p

p

p

p

p

p

p

p

p

p

p

NCb (+1 mg/l 2,4-D)

±

±

±

±

±

±

±

±

±

±

±

±

±

±

±

±

±

±

±

±

Shoot (+0.7 M sucrose, 4 weeks)

p

p

p

±

±

±

p

±

p

p

±

±

±

p

±

±

±

p

±

±

Shoot (+0.3 M NaCl, 4 weeks)

p

p

p

±

±

±

±

±

±

p

±

±

±

p

±

±

±

p

±

±

Shoot (+0.6 mM CdCl2, 4 weeks)

±

±

±

±

p

p

p

±

±

p

±

p

±

±

p

±

±

p

±

±

Shoot (+10 ± 4 M ABA, 2 weeks)

±

p

p

±

±

±

±

±

p

p

±

±

±

±

±

±

±

p

±

±

Shoot (+1 mg/l 2,4-D, 4 weeks)

p

p

p

p

±

±

±

±

p

p

p

±

p

±

±

p

±

p

p

p

Shoot (no additions, 2 Weeks)

p

p

p

p

p

±

p

±

±

±

p

±

±

±

±

±

p

p

±

±

Shoot (no additions, 3 days)

p

p

p

±

p

±

±

±

±

±

±

±

±

±

±

±

±

p

p

±

Table 1 continued Cell type or treatment

Phosphoprotein (spot no.)c 21

22

23

24

25

26

27

28

29

30

31

EC (+1 mg/l 2.4-D)

p

p

p

p

p

p

p

p

p

p

p

31/165(d)

NCb (+1 mg/l 2,4-D

±

±

±

±

±

±

±

±

±

±

±

10/157(d)

Shoot (+0.7 M sucrose, 4 weeks)

p

±

±

p

±

p

±

p

p

p

p

15/98(d)

Shoot (+0.3 M NaCl, 4 weeks)

p

±

±

p

±

p

±

p

p

p

p

13/103(d)

Shoot (+0.6 mM CdCl2, 4 weeks)

p

±

p

±

±

p

p

p

p

±

±

13/116(d)

Shoot (+10 ± 4 M ABA, 2 weeks)

p

±

±

p

p

p

±

p

p

p

p

13/110(d)

Shoot (+1 mg/l 2,4-D, 4 weeks)

p

±

±

±

p

p

p

p

p

p

p

20/150(d)

Shoot (no additions, 2 Weeks)

p

±

±

±

p

p

±

p

p

p

±

15/112(d)

Shoot (no additions, 3 days)

p

±

±

±

±

±

±

±

±

±

±

18/108(d)

a

a

Maintained for 3 months with biweekly subculture Maintained for 2 years with biweekly subculture c p, presence of phosphoprotein; ±, absence of phosphoprotein b

Discussion In order to gain a better understanding of the role of protein phosphorylation in the induction of embryogenic competence, we examined the phosphoproteins in EC, NC and shoot apices using chemical stress to

d

First number of ratio (in bold) is the number of phosphoproteins found in EC but not in NC; second number of ration is the number of phosphoproteins detected on gels shown in Figs. 1 and 2

induce somatic embryogenesis in carrot. We were able to determine that a particular phosphoprotein (no. 10) with a molecular mass of approximately 44 kDa was specific to EC and all of the stress treatments that induced embryogenic competence (Fig. 2, Table 1) but not to NC. The phosphorylation test showed that ECPP-44 was also phosphorylated in torpedo-shaped

745 Fig. 3A±F Patterns of two-dimensional fractionation of proteins in EC, NC and a control culture (shoot apices cultured for 2 weeks without stress compounds). A EC, B NC, C,D,E,F shoot apices in control culture. Arrows indicate silver-stained/ phosphorylated proteins that were consistently detected in all of the cells (A,B) and tissues (C,E; E is an enlarged view of part of C) examined. Circles (p) with open arrows (C) indicate the position of ECPP-44. D shows the original autoradiogram after fractionation of a sample that contained 300,000 cpm, and F is a magnified view of part of D. Square boxes (m) with arrows (C) indicate the presence of ECPP-44 in E and the absence of phosphorylation of this protein in F (shoot apices in control culture). Aliquots of 100 mg of protein were loaded on each gel |

|

746

Fig. 4 Peptide mapping and the autoradiograms of the peptides generated from phosphoprotein no. 10 (ECPP-44). The spot corresponding to phosphoprotein no. 10 (ECPP-44) in EC (see Fig. 3A) was obtained as described in Materials and methods. Some of the resultant bands of peptides (ec-1, ec-2, ec-3 and ec-4) were subjected to amino acid sequencing. Arrows (B) indicate the positions of peptides that were phosphorylated and comigrated with silver-stained peptides (ec-1 and ec-2). The amino acid sequences on the right represent those of each individual peptide

somatic embryos. Of particular interest was our observation that the ECPP-44 protein was not phosphorylated in the control cultures (shoot apices without stress treatment) although it was present at low levels (Fig. 3E, F). This was reconfirmed in recent preliminary works using specific ECPP-44 antibody against ECPP44 polypeptide in a Western blot analysis and immunoprecipitation assay. The antibody recognized the 44-kDa polypeptide in EC, somatic embryos, stress- and no stress-treated shoot apices and tap roots but not in NC. However, no phosphorylated-statue was detected in non-embryonic shoot apices (no Fig. 5 Nucleotide and deduced amino acid sequence of the cDNA fragment of ECPP-44. The amino acid sequence of the putative coding regions is shown beneath the nucleotide sequence. Regions conserved among the Group II LEA proteins, namely serine clusters, are double-underlined. Arrows above the sequences denote degenerate primers and a comparison of the internal peptide sequences of ECPP-44 (ec-1, ec-3 and ec-4) with parts of the predicted amino acid sequences of ERD10 and ERD14. Circles indicate amino acid residues that are identical in ECPP-44 and ERD10 or ERD14

stress-treated), tap roots and NC that did not have the ability to form somatic embryos by the immunoprecipitation assay (unpublished data). These results imply that the antigen is a dehydrin-like ECPP44 protein which might act in some embryonic roles via stress signals results for regulating carrot somatic embryogenesis. They also suggest that phosphorylation of ECPP-44 might be important for the acquisition of embryogenic competence. The amino acid sequence of the ec-3 fragment of ECPP-44 included a lysine-rich sequence (EKKGLKEKIEEK) that is characteristic of dehydrin-like proteins. Some dehydrins have a stretch of clusters of serine residues before the lysine-rich consensus sequence, and the serine residues in such clusters are phosphorylated by protein kinase CK2 (Plana et al. 1991). The ECPP-44 protein might have a stretch of such clusters before the EKKGLKEKIEEK sequence, and the serine residues in such clusters might be phosphorylated in vivo. We found that the ec-1 fragment could be phosphorylated, while the ec-3 fragment could not (Fig. 4). Partial sequencing of the cDNA fragment (141-bp; Fig. 5) suggested that the

747

ec-1 fragment was followed by the clusters of serine residues which could be potentially phosphorylated in vivo and then the ec-3 fragment. The sequence EKKGLKEKIEEK has been found in the deduced amino acid sequences of most homologs of RAB, a LEA protein, identified in other plant species (Close et al. 1993). This motif shows some degree of similarity to a sequence in a nuclear localization sequence (NLS) found in yeast mating-type factor a2 that functions in nuclear transport (Hall et al. 1984). Thus, ECPP-44 might be a regulator in the transduction of stress signals. In conclusion, we found ECPP-44 to be present under stress treatments capable of inducing embryogenic competency. While a minimum level of ECPP-44 was detected in the controls, phosphorylation experiments showed conclusively that it is phosphorylated during the transition to embryogenesis. Acknowledgements This work was supported in part by a Grant-in-Aid for Special Research Areas (Genetic Dissection of Sexual Differentiation and Pollination Processes in Higher Plants) from the Ministry of Education, Science, Culture and Sports, Japan; by a Grant-in-Aid from the "Research for the Future" Program of the Japan Society for the Promotion of Science; by the Special Coordination Funds of the Science and Technology Agency of the Japanese Government; and by the Program for Promotion of Basic Research Activities for Innovative Biosciences.

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