Tissue-specific expression of Pa18, a putative lipid ... - Springer Link

Plant Molecular Biology 42: 461–478, 2000. © 2000 Kluwer Academic Publishers. Printed in the Netherlands.

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Tissue-specific expression of Pa18, a putative lipid transfer protein gene, during embryo development in Norway spruce (Picea abies) Izabela Sabala1,3 , Malin Elfstrand1 , Isabelle Farbos2 , David Clapham1 and Sara von Arnold1,∗ 1 Uppsala

Genetic Center, Department of Forest Genetics, Swedish University of Agricultural Sciences, PO Box 7027, S-750 07 Uppsala, Sweden (∗ author for correspondence); 2 Uppsala Genetic Center, Department of Plant Biology, Swedish University of Agricultural Sciences, PO Box 7003, S-750 07 Uppsala, Sweden; 3 present address: Gene Expression Laboratory, Department of Molecular and Structural Biology, Aarhus University, 8000 Aarhus, Denmark Received 23 April 1999; accepted in revised form 15 November 1999

Key words: conifer, lipid transfer protein, Picea abies, somatic embryogenesis

Abstract A full-length Picea abies cDNA clone Pa18, encoding a protein with the characteristics of plant lipid transfer proteins, has been isolated and characterized. The size of the deduced 173 amino acid (aa) long protein is around 18 kDa. The first 100–120 aa show similarity to angiosperm lipid transfer proteins in amino acid sequence as well as in predicted secondary structure. The Pa18 gene is constitutively expressed in embryogenic cultures of Picea abies representing different stages of development as well as in non-embryogenic callus and seedlings. The Pa18 gene product has an antimicrobial activity. In situ hybridization showed that the Pa18 gene is equally expressed in all embryonic cells of proliferating embryogenic cultures but during embryo maturation the expression of the gene in maturing and mature somatic as well as in mature zygotic embryos is stronger in the outer cell layer than in other tissues. Southern blot analysis at different stringencies was consistent with a single gene with one or two copies rather than a gene family. Twenty independent transgenic sublines over- and under-expressing the Pa18 gene under the Zea mays ubiquitin promoter were established. There was a high yield of mature somatic embryos with a smooth surface only in untransformed, control cultures. Irrespective of the expression level of Pa18, the somatic embryos started to mature when given a maturation treatment. However, in the transgenic sublines, the outer cells in the maturing embryos frequently became elongated and vacuolated instead of remaining small and uniform. One explanation for this was that the expression of Pa18 was not restricted to the outer cell layer in transformed sublines. Angiosperms and gymnosperms separated about 300 million years ago and the embryo genesis is different in the two groups. The outer cell layer (protoderm), the first tissue to differentiate, is less clearly delineated in gymnosperms. For normal embryo development in angiosperms, expression of the LTP gene must be restricted to the protodermal cells. In this work we show that the expression of the Pa18 gene must be restricted to the putative protodermal cells of the gymnosperm.

Introduction During embryogenesis the zygote undergoes a complex series of morphological changes. Although the general plan of embryo development is similar in both angiosperms and gymnosperms, many differences can The nucleotide sequence data will appear in the DDBJ, EMBL and GenBank nucleotide sequence databases under the accession number AB007843.

be found in the pattern of embryo development between these two groups of plants. The first tissue to differentiate during embryogenesis in angiosperms is the protoderm which is formed by periclinal divisions of cells of the early globular embryo. The formation of the protoderm, which restricts cell expansion, is essential for the remaining developmental process (Dodeman et al., 1997). In contrast, in gymnosperms the cells in the surface layer of the em-

462 bryonal mass continue to divide periclinally as well as anticlinally, preventing differentiation of a distinct protoderm (Singh, 1978). In this study we address the question of whether the surface cell layer in the embryonal mass in gymnosperms corresponds to the protodermis in angiosperms. The Arabidopsis thaliana lipid transfer protein (LTP) gene (Atltp) has been shown to be expressed in the protoderm at the early globular stage of Arabidopsis embryo development (Vroemen et al., 1996). Furthermore, the Daucus carota ltp gene, EP2, is already expressed after five to six cell divisions in zygotic embryos and in precursor cell clusters from which somatic embryos develop (Sterk et al., 1991). Expression of the gene is then restricted to the protoderm cells. In Daucus carota ts11 mutant embryos, which are arrested at the late globular stage and lack a fully formed protoderm, the ltp gene is expressed in the subepidermal cell layer rather than in the protoderm (Sterk et al., 1991). Taken together, a correct expression pattern of ltp genes is required for normal embryo development in angiosperms. LTPs have been isolated from animals, fungi, plants and bacteria and are characterzed by their ability to catalyse the exchange of lipid molecules between artificial and natural membranes in vitro (Kader, 1996). LTPs are small, basic proteins ranging in size from 7 to 12 kDa, and share 30–70% similarity with each other (Yamada, 1992). The polypeptide chain contains eight cysteine residues at conserved positions allowing the formation of four disulfide bridges. LTPs are also characterized by a lack of tryptophan (Kader, 1996). Genes encoding LTPs have been isolated and characterized from various angiosperm species (Sossountzov et al., 1991; Sterk et al., 1991; Skriver et al., 1992; Thoma et al., 1994). LTPs are synthesized as precursors with a putative signal peptide (Bernhard et al., 1991) and are located extracellularly or are associated with the cell wall (Thoma et al., 1994; Molina et al., 1996); the expression patterns for different plant ltp genes are complex and, in many cases, temporally and spatially controlled. Moreover, each member of the ltp gene family can display its own peculiar expression pattern. In general, the expression of ltp genes is highest in young tissues especially in outer cell layers, mostly in the aerial parts of the plants (Sossountzov et al., 1991; Sterk et al., 1991; Thoma et al., 1994). However, Choi et al. (1996) have isolated a root-specific ltp from Phaseolus vulgaris. The functions of the LTPs have been widely discussed. One suggestion (Sterk et al., 1991) is that LTPs are involved in secretion or deposition of extra-

cellular lipophilic material, such as cutin monomers. Plant epidermal cells have an inner (basolateral) and an outer (apical) surface covered with a cuticle. The main components of cuticle are cutin monomers which are transported by polarized transport involving an epidermis-specific LTP residing in the outer cell wall (Thoma et al., 1993). This process would lead to the formation of a protective layer on the aerial parts of plants and around the young embryo which serves in protection against water loss (Sterk et al., 1991). LTPs have also been found to be involved in adaptation of plants to various environmental conditions, such as high and low temperature or drought (Keresztessy and Hughes, 1998; Trevino and O’Connell, 1998). Some of the characterized LTPs are induced by abscisic acid (ABA) or salicylic acid which are known to mediate plant responses to environmental stresses (Hughes et al., 1992; Hollenbach et al., 1997). Several LTPs have been characterzed by their antimicrobial activity, such as Ace-AMP1 from Alium cepa (Cammue et al., 1995). Ace-AMP1 had a strong action on gram-positive bacteria but no effect on gramnegative bacteria in vitro. Another protein, LTP2 from Hordeum vulgare, has been shown to be effective against the bacterial pathogens Clavibacter michiganensis ssp. sepedonicus (Caaveiro et al., 1997) and Pseudomonas syringae (Molina et al., 1997). Overexpression of the ltp2 gene from Hordeum vulgare in Nicotiana tabacum and Arabidopsis thaliana enhanced tolerance to bacterial pathogens; furthermore, growth of Pseudomonas syringae was more retarded on the leaves of transgenic N. tabacum plants than on control leaves (Molina et al., 1997). Terras et al. (1992) have proposed a model in which LTPs play a role in defence. According to this model LTPs could confer defence in two different ways: indirectly by their involvement in the formation of a mechanical cutin barrier and directly by their intrinsic antifungal activity upon deposition of the transported cutin monomers. As part of our research programme on somatic embryogenesis in conifers we are studying the regulation of early embryo development in Picea abies. Somatic embryos of conifers have been studied for several years with P. abies as a model system (von Arnold et al., 1995). These studies have shown that somatic embryos from different embryogenic cell lines exhibit different capacities to proceed through the whole developmental pathway. Proliferating embryogenic cultures of P. abies consist of single cells, proembryogenic masses (PEMs) and somatic embryos (Filonova

463 et al., 1998). According to morphology, maturation capacity and secretion of proteins, the embryogenic cell lines have been divided into two groups, A and B (Jalonen and von Arnold, 1991; Egertsdotter and von Arnold, 1993). Group A cell lines develop mature somatic embryos after treatment with ABA. In group B cell lines the somatic embryos are considered to be developmentally blocked in the sense that they cannot reach the developmental stage required for response to ABA and formation of mature somatic embryos. The critical developmental stage required for response to ABA is characterized by well developed somatic embryos having a distinct, dense and large embryonic region (Egertsdotter and von Arnold, 1995). In contrast, in group B cell lines the somatic embryos are less developed, with a smaller and loosely packed embryonic region. In order to determine if tissue specification occurs in the embryonic region of somatic embryos of Picea abies comparable to the differentiation of protoderm in angiosperms, we have isolated an ltp-like gene which is expressed in somatic embryos of P. abies. During development of somatic embryos there is a switch in the expression pattern of the gene. A correct expression pattern of the gene is required for normal embryo development. Our data demonstrate that differentiation of a surface cell layer occurs early during embryo development in gymnosperms.

Materials and methods Plant materials Embryogenic cell lines of Picea abies (L.) Karst were initiated from mature zygotic embryos and established as described elsewhere (Egertsdotter and von Arnold, 1993). Each cell line represents one genotype. The cell lines were grown as suspension cultures in proliferation medium, i.e. half-strength LP medium (von Arnold, 1987) with 9.0 µM 2,4-D and 4.4 µM BA. Each 500 ml Erlenmeyer flask contained 100 ml suspension cultures. The cultures were incubated in darkness at 20 ◦ C and were subcultured every week. Embryogenic cell lines representing type A (A66, A37) and type B (B1, B41) were used in this study. Non-embryogenic callus was initiated from zygotic embryos and grown as described previously (Egertsdotter and von Arnold, 1995). The nonembryogenic calli were grown in petri dishes containing proliferation medium and solidified with 0.18%

gellan gum (Merck). The cultures were incubated in darkness at 20 ◦ C and subcultured every month. Seeds of Picea abies were surface-sterilized by continuous shaking for 25 min in 7.5% w/v calcium hypochlorite and 0.3% v/v Tween 20 and then rinsed three times in sterile water. The seeds were soaked in sterile water for 21 h at 4 ◦ C and sown and germinated in Magenta jars containing a sterile mixture of vermiculite and water. The seedlings were grown for one month in a culture room at 22 ◦ C under continuous light from white fluorescent tubes (20–30 µmol m−2 s−1 ). cDNA library screening A cDNA library was constructed from mRNA isolated from ABA-treated, type A embryogenic cultures of P. abies as described previously (Sabala et al., 1997). A Pinus taeda LTP cDNA clone (Kinlow et al., 1994) labelled with 32 P was used to screen ca. 35 000 plaques of a λgt11 cDNA library constructed from mRNA isolated from an ABA-treated, A66 embryogenic cell line of P. abies. The library screening procedure has been described previously (Sabala et al., 1997). Two plaques giving positive signals were purified and designated pltp11.1 and pltp23.2. The pltp23.2 insert was cloned into the NotI site of Bluescript SK+ plasmid. To confirm that the isolated clone was not chimeric, RT-PCR was performed with total RNA from P. abies seedlings that had been treated with DNase to remove residual DNA. For PCR reactions two insert specific primers ltp-1 and ltp-2 located in the end and in the beginning of the open reading frame were used. The PCR product was sequenced. DNA sequencing and sequence analysis The nucleotide sequence was determined using thermal cycle sequencing with dye terminators and the ABI automatic sequencer (ABI, Applied Biosciences, Foster City, CA). The sequencing of 50 and 30 ends of pltp11.1 and pltp23.2 inserts was done with phage DNA and λgt11 reverse and forward primers. The complete sequence of pltp23.2 insert was obtained after cloning into the plasmid and using Bluescript specific primers and the insert-specific primer. RNA extraction and northern blot analysis Total plant RNA was isolated as described previously (Sabala et al., 1997). For northern analysis

464 10 µg of total RNA was separated by gel electrophoresis and blotted onto a Hybond-N+ nylon membrane (Amersham, Buckinghamshire, UK). The insert was multiplied by PCR with Bluescript primers, gelpurified with the Geneclean II kit (BIO 101, Vista, CA) and labelled with 32 P-dCTP by means of the oligolabelling kit (Pharmacia, Uppsala, Sweden). Hybridization was performed overnight at 42 ◦ C in 5×SSC, 4× Denhardt’s solution, 0.1% SDS, 40% formamide, 10% dextran sulfate and 100 µg/ml denatured salmon sperm DNA. The membranes were washed in 2×SSC, 0.1% SDS (2 × 15 min at room temperature) and in 0.2× SSC, 0.1% SDS (2 × 15 min at 37 ◦ C and at higher temperatures when necessary). In situ hybridization Proliferating, maturing and mature somatic embryos from cell line A66 and from transformed sublines over- and under-expressing the Pa18 gene as well as zygotic embryos were analysed by in situ hybridization. The sense and antisense RNA probes (entire Pa18 gene) were labelled with digoxygenin by using the DIG RNA Labeling Kit (Sp6/T7) (Boehringer Mannheim, Mannheim, Germany). The tissues were fixed in formalin-acetic acid solution and hybridized according to De Block and Debrouwer (1996) with proteinase K treatment shortened to 15 min and levamisol (1 mM) added to the colour solution. Extraction of genomic DNA and Southern blot analysis Total DNA was isolated from embryogenic cultures of Picea abies by the method of Bernatzky and Tanksley (1986) with modifications as described (Yibrah et al., 1996). 32 µg of total DNA was digested with EcoRI, HindIII, BamHI, SacI or KpnI, separated on 0.8% agarose gel and transferred onto Hybond-N+ nylon membrane, essentially as described by Sambrook et al. (1989). The pltp23.2 probe was prepared as for northern blot analysis and ltp cDNA from Pinus taeda was prepared as for cDNA library screening. The filters were washed according to Sambrook et al. (1989). ABA and cold treatments In order to study if ABA treatment increases expression of Pa18, embryogenic suspension cultures and one-month-old seedlings were treated with ABA (± cis-trans isomer, Sigma, St. Louis, MO) as described

before (Sabala et al., 1996; 1997). Embryogenic cultures were treated with ABA which was added to a final concentration of 15 µM for 44 h. Seedlings were sprayed with a 15 µM ABA solution and harvested after 44 h. For cold treatment the seedlings were incubated in continuous light (20–30 µmol m−2 s−1 ) at 4 ◦ C for 44 h. Filtered embryogenic cultures, whole seedlings and seedlings dissected into roots, hypocotyls, epicotyls and cotyledons were frozen in liquid nitrogen and stored at −80 ◦ C. Transformation of somatic embryos In order to study the effect of under- and overexpression of Pa18 gene on embryo development, two embryogenic cell lines (A66 and B1) were transformed with the Pa18 gene in sense orientation driven by the Zea mays ubiquitin promoter. The modified pAHC25 plasmid containing the bar gene and cut with SmaI and SacI enzymes to remove the uidA cassette was used for transformation (Christensen and Quail, 1996). The Pa18 insert was first cloned into the NotI site of Bluescript SK+ and then cut with SmaI and SacI enzymes and moved to modified pAHC25 plasmid. A66 cultures were also transformed with the unmodified plasmid pAHC25. Embryogenic cultures were transformed by particle bombardment using a particle inflow gun (Finer et al., 1992) as modified by Professor H.-U. Koop (Clapham et al., 1999). Gold particles (1.5–3.0 µm diameter, Aldrich) were coated by suspending 10 mg in 210 µl water in an Eppendorf tube, adding 20 µg of plasmid DNA in 10 mM Tris, 1 mM EDTA buffer, pH 8.0, with vortexing followed rapidly by one tenth volume of 3 M sodium acetate and 2.5 volumes ethanol. The suspension was placed at −20 ◦ C for 30 min, and centrifuged briefly, after which the supernatant was removed and replaced with 1 ml ethanol. Cell samples (100–150 mg fresh weight) taken 4– 6 days after subculture were spread in thin layers on the surface of sterile filter paper discs (Clapham et al., 1995). Discs with cells were placed on filter paper beds soaked in proliferation medium containing 0.25 M myoinositol for 1–3 h before bombardment. A partial vacuum equivalent to about 10 kPa was applied to the cells just before bombardment. To select for stable transformants, the filter paper discs with bombarded cells were transferred to solid proliferation medium containing 0.25 M myoinositol and 0.18% gellan gum directly after bombardment.

465 On day 8, the cells were transferred to proliferation medium containing 5-azacytidine (3 mg/l) and Basta (Hoechst) at 1 mg Basta solution per litre in addition to myoinositol. On day 15, the cells were transferred to proliferation medium with Basta and myoinositol. They were subcultured on this medium monthly. Embryogenic callus resistant to Basta appeared from 2–4 months after bombardment. When the calli were about 2 mm in diameter they were subcultured onto standard proliferation medium without Basta or extra myoinositol. The selected embryogenic cultures continued to proliferate and gave rise to sublines within 1–2 months. To confirm that the selected sublines were transgenic, northern blots were probed with the bar gene. Analysis of transformants Twenty sublines transformed with the sense construct were recovered and analysed. The level of Pa18 transcript in transformed lines was determined by northern analysis with 10 µg of total RNA and full-length insert as a probe. For an internal control the same membranes were stripped and reprobed with an 18S probe. The intensity of signals was quantified with the ImageQuant software for PhosphoImager (Molecular Dynamics). The morphology of PEMs in proliferating cultures was analysed. For statistical analysis transformation of the data was performed. The percentage of PEMs with vacuolated, elongated cells was transformed as follows: V = ln (% PEMs with vacuolated, elongated cells /% PEMs without elongated vacuolated cells). Pa18 expression was transformed as follows: Y = 1/rt Pa18 expression. Regression analysis (with Statistica 4.1, Statsoft, Tulsa, OK) was carried out to investigate if there was a correlation between the morphology of PEMs and the expression of Pa18 in the sublines. Maturation of somatic embryos from the transformed sublines was performed as described previously (Bozhkov and von Arnold, 1998). Embryogenic cultures proliferating on solidified proliferation medium were suspended in liquid half-strength LP medium lacking plant growth regulators. After one week’s pre-treatment, the cultures were plated on filter papers placed on maturation medium, i.e. BMI-SI medium containing 90 mM sucrose, 24 µM ABA, 7.5% PEG, 450 mg/l glutamine and solidified with 3.5 g/l gelrite. Maturation was tested in two independent experiments.

Testing for antimicrobial activity Four sublines of the embryonic cell line A66 showing different expression of the Pa18 and untransformed A66 were chosen for studying antimicrobial activity. Embryogenic cultures (ca. 500 mg) were resuspended in proliferation medium supplemented with 450 mg/l glutamine and grown for 7 days on a rotary shaker. The cultures were immobilized in proliferation medium supplemented with glutamine (450 mg/l) and solidified with agarose (0.6% LMP). Equal volumes of suspension and proliferation medium supplied with 1.2% LMP agarose were mixed and 1 ml of the mixture was plated in each 6 cm petri dish. The immobilized cultures were incubated for 5 days before they were overlaid with Agrobacterium tumefaciens. A. tumefaciens (strain C58) was grown overnight in LB medium at 28 ◦ C on a rotary shaker (200 rpm). Cells were collected by centrifugation (3500 rpm for 15 min at room temperature) and suspended in proliferation medium supplemented with glutamine (450 mg/l). The resuspended bacteria were diluted to a density corresponding to 0.1 or 0.2 at 600 nm (OD600 0.1 or 0.2). The bacterial suspensions were mixed with an equal volume of proliferation medium supplemented with glutamine (450 mg/l) and agarose (1.2% LMP), giving an OD600 of either 0.05 or 0.1. Of the mixture 1 ml was spread on each plate with immobilized embryogenic cultures. Ten plates of each subline and bacterial concentration were prepared. The plates were inoculated in darkness at 24 ◦ C and then analysed after 20 h. The experiment was repeated. The plates were separated into four fields (A–D) and all PEMs or aggregates of PEMs in each field were classified either as PEMs with a clearing zone (inhibition of bacterial growth) or PEMs without a clearing zone. The fields were summed and the percentage of PEMs with a clearing zone was calculated. Logistic transformation of the data was performed (logistic variable = ln (%zones/% no zones)). Regression analysis (with Statistica 4.1, Statsoft, Tulsa, OK) was carried out to investigate if there was a correlation between the formation of clearing zones and the expression of Pa18 in the sublines.

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Figure 1. Nucleotide sequence and deduced amino acid sequence of the Pa18 cDNA clone. The predicted signal peptide is underlined, the protein sequence designating the putative ATP/GTP binding site motif is bolded, the putative phosphorylation sites are indicated by an asterisk and the putative glycosylation sites are marked with #. The stop codon is bolded and underlined. The nucleotide sequence data will appear in the DDBJ, EMBL and GenBank nucleotide sequence databases under the accession number AB007843.

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Figure 2. Similarity between amino acid sequences of PA18 and LTPs. The sequences of signal peptides were not aligned. The LTP sequences used in the alignment have the following accession numbers in the GenBank database: Picea abies AB007843; Pinus taeda, U10432; Daucus carota, M64746; Nicotiana tabacum, D13952; Hordeum vulgare, P20145; Senecio odorus, L33791.

Results Isolation and characterization of Pa18 Screening of the cDNA library resulted in the recovery of two positive plaques, pltp11.1 and pltp23.2, from among ca. 35 000 screened. The inserts pltp11.1 and pltp23.2 were about 500 and 800 bp long, respectively. The pltp23.2 clone showed similarity to the non-specific lipid transfer protein genes found in the databases and was chosen for further characterization. The pltp11.1 clone could not be identified by sequence similarity to any previously determined DNA or protein sequences and was not characterized further.

The pltp23.2 clone was completely sequenced and found to consist of 795 bp with one 519 bp long open reading frame which begins 12 bp from the 50 end and terminates at 531 (Figure 1). There are several ATG codons in the beginning of the open reading frame. The flanking region of the ATG codon at position 12– 14 has an optimal flanking sequence for eukaryotic translation initiation, G at position 15 and A at position 9 (Gallie, 1993) and this codon is the most likely start codon. No poly(A) tail or polyadenylation signals are present at the 30 end of the clone. The open reading frame predicts a 173 aa long polypeptide with a calculated molecular mass of 17 963 Da. The deduced protein was designated PA18 and the clone Pa18. The amino acid sequence is serine-rich (15.6%) and contains 11 cysteines. This protein lacks tryptophan and

468 quence. The amino acid sequence 110–117 has characteristics of the ATP/GTP-binding motif A present in various groups of proteins, including several proteins involved in active transport (Saraste et al., 1990). The function of transferring phospholipids is revealed in the secondary and tertiary structure of LTPs (Désormeaux et al., 1992; Ostergaard et al., 1995). The Protein Analysis program estimated the secondary structure of the PA18 based on the amino acid sequence. The results presented in Figure 3B showed the protein to be 46% helical. Within the region of similarity to LTPs (first 120 aa) the percentage of helical structures increases to 60%. Four separate regions with helical structure were also predicted in the N-terminus of the PA18 while the C-terminus, from about amino acid 110, has an extended secondary structure. The putative transmembrane helix was predicted between amino acids 148 and 165. Pa18 is not a member of a multigene family Figure 3. A. Hydropathy plot of the deduced amino acid sequence of the PA18 protein. The hydropathy plot was predicted according to the method of Kyte and Doolitte (1982) using a window of 7 amino acids. B. Prediction of the secondary structure of PA18 protein. H, helix, E, extended; C, coil.

histidine. As shown in Figure 2, the deduced protein sequence shows similarity to several non-specific lipid transfer proteins. The positions of the cysteines correspond to the highly conserved cysteine positions in plant LTPs. Since this similarity was observed only within the first 100–120 aa, RT-PCR reactions were performed to check whether the isolated clone was chimeric. A fragment of the expected size (500 bp) was produced in the reaction with primers located in the beginning and in the end of the open reading frame. The sequence of the PCR product showed that the pltp23.2 clone is not chimeric. The hydropathy plot of the deduced protein, PA18, revealed that the first 25 aa constitute a hydrophobic region typical of a eukaryotic signal peptide (Figure 3A). The most likely cleavage site is between amino acids 25 (Cys) and 26 (Glu) (von Heijne, 1986) (Figure 1). Using the PROSITE program of the GeneWorks (IntelliGenetics, Mountain View, CA) sequence analysis software, several putative motifs were found within the PA18 protein sequence. Three protein kinase phosphorylation sites were predicted in the mature PA18 protein (Figure 1). There are also four putative glycosylation sites present within the PA18 protein se-

To investigate if Pa18 belongs to a gene family, Southern blot analysis was performed. Five different enzymes, EcoRI, HindIII, BamHI, SacI and SpeI, were used to cleave genomic DNA. Since none of these enzymes cuts the Pa18 gene, a single band was expected if the investigated gene is the only member of the gene family. As shown in Figure 4, single bands were obtained in all digestions with high-stringency washing. When filters were washed at low stringency one weaker band was observed in EcoRI and SacI digestions. Genomic DNA from Picea abies, digested with EcoRI and HindIII, was also probed with the ltp cDNA clone from Pinus taeda that has been used for screening the P. abies cDNA library. Similar results were obtained as for Pa18 as shown in Figure 4. The copy number reconstruction confirmed that there are 1 or 2 copies of the Pa18 gene in the P. abies genome (data not shown). Tissue-specific expression pattern of Pa18. The expression of the Pa18 gene was studied by northern blot analysis. Embryogenic cell lines of Picea abies representing group A (cell line A66) and group B (cell line B1) as well as non-embryogenic callus and roots, cotyledons and hypocotyls from 1-month old seedlings were analysed. As shown in Figure 5A, the Pa18 gene was expressed in both A and B groups of embryogenic cultures as well as in non-embryogenic callus. ABA treatment did not affect the expression of Pa18 in embryogenic cultures. Furthermore, the Pa18

469 gene was expressed in P. abies seedlings (Figure 5B). The expression was higher in hypocotyl and roots than in the cotyledons. In seedlings, cold and ABA treatment did not significantly affect the level of the Pa18 gene transcript but another abundant transcript with a size of 4 kb appeared after ABA treatment. Expression pattern of the Pa18 gene during embryo development

Figure 4. Southern blot analysis of Pa18 gene in Picea abies. Genomic DNA (40 µg) was cleaved with EcoRI, BamHI, HindIII, SacI or KpnI and probed with the entire Pa18 clone. The filters were washed at high stringency (0.2× SSC, 0.1% SDS at 60 ◦ C).

In situ hybridization was performed to localize the expression of Pa18 in proliferating and maturing embryogenic cultures as well as in mature somatic and zygotic embryos. This expression pattern is shown in Figure 6. There were no signals detected when the sense probe was used for hybridizations (Figures 6B, D, F, H, J, L, N). PEMs in proliferating embryogenic cultures of cell line A66 expressed the Pa18 gene in all embryonic cells (Figure 6A). The expression pattern was similar in PEMs from cell line B1 (data not shown). Pa18 was also expressed in all embryonic cells of somatic embryos in proliferating cultures (Figure 6C). During maturation of the somatic embryos the expression pattern of Pa18 successively changed from uniform expression in the cells of the embryonic region to stronger expression in the outer cell layer (Figures 6E, G). In mature somatic embryos Pa18 was strongly expressed in the outer cell layer (Figures 6I, K). Also in mature zygotic embryos, Pa18 was preferentially expressed in the outer cell layer (Figure 6M). Since vacuolated cells were destroyed during sample preparation it was difficult to draw any conclusions as to the expression of the gene in these cells. Expression of Pa18 in transgenic embryogenic cultures

Figure 5. Northern blot analysis of Pa18 expression in Picea abies. A. Expression of Pa18 in embryogenic cultures; 1, cell line A66; 2, cell line A66 treated with ABA (15 mM, 44 h); 3, cell line B1; 4, cell line B1 treated with ABA (15 mM, 44 h); 5, non-embryogenic callus. B. Expression of Pa18 in 1-month old seedlings: 1, hypocotyls; 2, cotyledons; 3, roots; 4, whole seedlings 44 h after spraying with ABA (15 mM); 5, whole seedlings after cold treatment (+4 ◦ C, 44 h). In each lane 10 µg of total RNA was loaded. Filters were probed with Pa18 and washed at moderate stringency (0.2× SSC, 0.1% SDS at 50 ◦ C).

Twenty sublines growing on BASTA media were isolated and analysed. Nineteen of the sublines gave positive results when checked for the presence of the bar transcript. The expression level of Pa18 was compared in different sublines when setting the expression level in the untransformed control to 1. However, it has to be stressed that the expression level of Pa18 was almost twice as high in B1 as in A66. Both under- and over-expression of Pa18 gene was detected among transformed sublines. The level of the Pa18 transcript varied from 0.5 to 8.1 times of the untransformed control (Figure 7). For sublines derived from the A66 line the level of the Pa18 transcript varied between 0.5 and 8.1 while for the B1 sublines this variation was much lower, between 0.5 and 3.2. In

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Figure 6. In situ localization of the Pa18 transcript at different stages of A66 somatic embryo development and in mature zygotic embryos. A–L. Somatic embryos from control cell line A66. M–N. Zygotic embryos. O. Embryo over-expressing the Pa18 gene, subline 1D. P. Embryo under expressing the Pa18 gene, subline 6C. A, B. PEMs in proliferating cultures. C, D. Somatic embryos in proliferating cultures. E, F. Maturing embryos after 3 weeks of maturation treatment. G, H. Maturing embryos after 6 weeks of maturation treatment. I, J. Cotyledonary stage somatic embryo. K, L. Mature somatic embryo. k. Higher magnification of the cotyledon from K. A, C, E, G, I, K, M, O, P. Hybridizations with antisense probe. B, D, F, H, J, L, N. Hybridizations with sense probe. Arrows indicate embryonic region. Scale bars represent 200 µm in A, B, C, D, E, F, G, H, O and P, 300 µm in I, J, K, L, M and N, and 100 µm in k.

the subline A66 9A, in which the bar transcript was not detected, the level of Pa18 transcript was similar to that of the untransformed control. According to the level of Pa18 expression, the sublines have been divided into three groups: group I with expression lower than control (0.5–0.6), group II with expression corresponding to expression in control (1.1–1.9) and group III with enhanced expression (2.5–8.1). The northern blot analysis was repeated. The expression of the Pa18 varied slightly between the two independent measurements; however, the classification of the sublines into groups was the same.

All transformed sublines have been analysed regarding embryo morphology and their ability to mature after treatment with ABA. The control suspension cultures of lines A66 and B1 contained small aggregates. The appearance of suspensions was the same for B1 sublines irrespective of whether they over- or under-expressed Pa18. In contrast, sublines of line A66 overexpressing Pa18 formed large aggregates in suspension cultures, while the underexpressing sublines formed smaller aggregates as compared to the control (Figures 8A, D, G). Proliferating embryogenic cultures of Picea abies consist of single cells, PEMs and somatic embryos.

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Figure 7. Expression of Pa18 in transformed embryogenic sublines of Picea abies. Embryogenic cell lines A66 and B1 were transformed with the Pa18 gene fused to the ubi promoter. Stably transformed sublines were established. Northern blot analysis was performed as described in Materials and methods. To check the amount of RNA loaded filters, were hybridized with an rRNA 18S probe. The quantification of signals was performed with the ImageQuant software for the PhosphoImager. The expression level of the control cell lines A66 and B1 was set at 1.

In a tracking study we have followed the development of thousands of single cells, PEMs and somatic embryos in proliferation medium (Filonova et al., 1998). The PEMs develop from small cell aggregates into large clusters, with vacuolated and meristematic cells from which somatic embryos differentiate. A proliferating culture is mainly composed of PEMs at various stages of development. No changes in morphology of PEMs were observed in sublines of line B1. However, in transformed sublines of line A66 the PEMs had a changed morphology. PEMs in the control A66 cell line consisted of elongated and non-elongated vacuolated cells as well as meristematic cells (Figure 8B). In sublines under-expressing Pa18 the PEMs lacked elongated, vacuolated cells (Figure 8E) while in sublines over-expressing Pa18, the distinct embryonic region is surrounded by vacuolated cells which are highly elongated (Figure 8H). A highly significant positive correlation was found between expression level of Pa18 and the presence of elongated, vacuolated cells in PEMs r 2 = 0.65 P < 0.001); also see Table 1. The ability to form maturing somatic embryos when treated with ABA was determined for all transformed sublines. Cell line B1 will normally not form mature somatic embryos. Neither did any transformed B1 subline respond to ABA. All B1 sublines, including the control, continued to proliferate on maturation medium. Cell line A66 will normally form mature somatic embryos on maturation medium. The transformed A66 sublines responded to maturation treatment but in a different way from the control. In the

Table 1. The expression of Pa18 in relation to the percentage of PEMs with vacuolated elongated cells present in proliferating cultures of A66 sublines. Subline

Pa18 expressiona

control 6D 6C 6E 8B 9A 6A 6B 10A 5B 1C 1A 1B 1D

1.0 0.5 0.6 0.6 0.6 1.0 1.1 1.3 1.4 1.9 2.5 5.2 6.9 8.1

% elongated vacuolated cellsb 45.3 17.4 9.2 20.8 13.3 24.5 30.2 6.9 26.5 38.3 75.7 60.2 80.3 72.0

a The level of Pa18 expression is presented in Figure 7. The expression of Pa18 in control A66 cell lines was set to 1. b The number of PEMs with vacuolated, elongated cells (Figures 8B and H) and without vacuolated, elongated cells (Figure 8E) were counted. At least 200 PEMs were counted per subline.

beginning of the maturation treatment, after about 4 weeks, many immature embryos with a distinct embryonic region and a well developed suspensor region were formed. At this developmental stage the Pa18 gene was preferentially expressed in the outer cell layer in the control embryos (Figure 6G) but evenly expressed in the embryonic region in both over-

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Figure 8. Morphological characterization of control cell line A66 and sublines under- and over-expressing Pa18. A. Overview of proliferating suspension culture of the control A66 cell line; note big PEM aggregates (a). B. Higher magnification of PEMs from A. C. Maturing somatic embryo of the control A66 cell line after 4 weeks in maturation medium; note smooth outer surface indicated by arrow. D. Overview of proliferating suspension culture of the under-expressing subline A66 6E; note small PEM aggregates (a). E. Higher magnification of PEMs from D. F. Maturing somatic embryo of the subline A66 6E after 4 weeks on maturation medium; note lack of smooth outer surface indicated by arrow. G. Overview of proliferating suspension culture of the overexpressing subline A66 1D; note large PEM aggregates (a). H. Higher magnification of PEM from G. I. Maturing somatic embryo of overexpressing subline A66 1D after 4 weeks on maturation medium; note lack of smooth outer surface indicated by arrow. a, PEM aggregates; m, meristematic cells; v, vacuolated cells; ve, vacuolated, elongated cells. Scale bars represent 1.5 mm in A, D and G, 200 µm in B, E and H, and 400 µm in C, F and J.

473 Table 2. Development of mature somatic embryos in transgenic sublines over- and under-expressing Pa18. Subline Expression Total Proportion (%) of embryos level number of Pa18 of embryos /g of tissue arresteda overgrownb normalc control 1B 1D 6C 6E

1.0 6.9 8.1 0.6 0.6

906 264 568 674 169

32 38 25 69 14

28 42 65 25 81

40 20 10 6 5

a Embryos arrested before the cotyledonary stage (Figure 9B). b Embryos overgrown by suspensors (Figure 9(C). c Normal mature embryos (Figure 9A).

expressing (Figure 6O) and under-expressing maturing somatic embryos (Figure 6P). In the control A66 cell line the embryonic region of the maturing somatic embryo usually had a smooth surface (Figure 8C). In contrast, the embryonic region of most of the maturing embryos in sublines under- and over-expressing Pa18 had an irregular surface (Figures 8F and J). The immature embryos either continued to develop into normal mature embryos (Figure 9A), were arrested at a developmental stage before the cotyledons were formed (Figure 9B) or became overgrown by suspensor cells (Figure 9C). All developmental pathways were observed both in controls and sublines. However, in the control, 40% of the maturing somatic embryos developed into normal mature embryos (Table 2). The yield of normal mature embryos was lower (P