Characterization of CsSEF1 gene encoding putative CCCH-type zinc finger protein expressed during cucumber somatic embryogenesis

ARTICLE IN PRESS Journal of Plant Physiology 166 (2009) 310—323

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Characterization of CsSEF1 gene encoding putative CCCH-type zinc finger protein expressed during cucumber somatic embryogenesis Agnieszka Grabowskaa,1, Anita Wisniewskab,,1, Norikazu Tagashirac, Stefan Malepszyd, Marcin Filipeckid a

Department of Biochemistry, Faculty of Agriculture and Biology, Warsaw University of Life Sciences, Nowoursynowska 159, 02-776 Warsaw, Poland b Department of Plant Physiology, Faculty of Agriculture and Biology, Warsaw University of Life Sciences, Nowoursynowska 159, 02-776 Warsaw, Poland c Faculty of Human Life Science, Hiroshima Jogakuin University, 4-13-1 Ushita-Higashi, Higashiku, Hiroshima city, 732-0063 Japan d Department of Plant Genetics Breeding and Biotechnology, Faculty of Horticulture and Landscape Architecture, Warsaw University of Life Sciences, Nowoursynowska 159, 02-776 Warsaw, Poland Received 20 February 2008; received in revised form 27 May 2008; accepted 2 June 2008

KEYWORDS CCCH zinc finger; Cotyledon primordium; Cucumber; Embryogenesis; Somatic embryo

Summary Somatic embryos obtained in vitro are a form of vegetative reproduction that can be used in artificial seed technology, as well as a model to study the principles of plant development. In order to isolate the genes involved in somatic embryogenesis of the cucumber (Cucumis sativus L.), we utilized the suppression subtractive hybridization (SSH). One of the obtained sequences was the CsSEF1 clone (Cucumis sativus Somatic Embryogenesis Zinc Finger 1), with a level of expression that sharply increased with the induction of embryogenesis. The full length cDNA of CsSEF1 encodes the putative 307 amino acid long protein containing three zinc finger motifs, two with CCCH and one with the atypical CHCH pattern. The CsSEF1 protein shows significant similarity to other proteins from plants, in which the zinc fingers arrangement and patterns are very similar. Transcripts of CsSEF1 were localized in the apical part of somatic embryos, starting as early as the polarity was visible and in later developmental

Abbreviations: ECS, embryogenic cell suspension; SE, somatic embryogenesis or somatic embryos; SSH, suppression subtractive hybridization; ZE, zygotic embryogenesis or zygotic embryo. Corresponding author. Tel.: +48 22 59 325 33; fax: +48 22 59 325 21. E-mail address: [email protected] (A. Wisniewska). 1 These authors shared equal participation in this work. 0176-1617/$ - see front matter & 2008 Elsevier GmbH. All rights reserved. doi:10.1016/j.jplph.2008.06.005

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stages marking the cotyledon primordia and procambium tissues. As a result of transferring an antisense fragment of CsSEF1 into Arabidopsis thaliana abnormalities in zygotic embryos and also in cotyledons and root development were observed. & 2008 Elsevier GmbH. All rights reserved.

Introduction Somatic embryogenesis (SE), the formation of somatic embryos from cells other than the zygote, most frequently under in vitro culture conditions, is an important process for biotechnology allowing for the effective clonal propagation of plants. SE can also serve as a model for studying the functions of genes involved in zygotic embryogenesis, since there are generally many fundamental similarities in the course of both processes (Dodeman et al., 1997). In dicotyledonous plants, the development of the somatic embryo usually involves the globular and heart/torpedo stages, which resemble the developmental stages of a zygotic embryo (Zimmerman, 1993; Mordhorst et al., 1997). Somatic embryos of the cucumber correspond to zygotic ones up to the heart stage, whereas later stages can differ, mainly in the structure of cotyledons (Tarkowska et al., 1994). Somatic embryogenesis as a model for studying genes involved development is of special importance in species other than Arabidopsis thaliana, due to lack of rich collections of embryonic mutants. Moreover, there are not many species that follow a ‘‘typical’’ Arabidopsis thaliana embryo development model (Kaplan and Cooke, 1997). Substantial differences can occur even during the first division of the zygote, which in most plants is transverse, but there are species in which the first division of the zygote is longitudinal or oblique. A dormancy of seeds, which is traditionally regarded as the end-point of embryogenesis in some plants, may not occur at all. In grasses, the embryo in the dormant stage already has leaf primordia and adventitious roots, whereas in orchids the development of the embryo is arrested at the globular stage (Kaplan and Cooke, 1997). For cucumber, two strategies aimed at obtaining embryogenic tissue have been developed: one is based on a synthetic auxin (2,4-dichlorophenoxyacetic acid) (Wro ´blewski et al., 1995), and the second involves the presence of a cytokinin in the medium (benzylaminopurine) (Burza and Malepszy, 1998). These methods maintain a cell suspension in the undifferentiated state (an embryogenic cell suspension, ECS) as a result of constant auxin or cytokinin pressure in the liquid medium. Transferring the culture to a medium lacking growth regulator initiates organized cell divisions that lead

to the formation of somatic embryos. These strategies for the somatic embryogenesis of cucumber have many advantages that favor molecular studies. These include a relatively short time for induction of ECS from primary explants, long-term maintenance of embryogenic potential by the ECS (for over 2.5 years), and easy observation and access to the individual developmental stages of the embryo (Burza et al., 2006). Only a few genes involved in somatic embryogenesis in the cucumber have been described thus far: CUS1, Cs-XTH1 and Cs-XTH3 (Filipecki et al., 1997; Malinowski et al., 2004). The largest number of genes participating in SE, over a dozen, has been characterized in carrot (for instance EMB-1 (Wurtele et al., 1993), CEM-6 (Sato et al., 1995), CHB (Kawahara et al., 1995; Hiwatashi and Fukuda, 2000), DcSERK (Shah et al., 2001) or KDC2 (Formentin et al., 2006)). For other species, there are only single examples. Recently, it was confirmed that the soybean orthologue of the Arabidopsis MADS-domain transcription regulator AGL15 is able to increase somatic embryo production (Thakare et al., 2008) and PaVP1 transcription factor can be regarded as a good marker of somatic embryogenesis in Norway spruce (Fischerova et al., 2008). In Medicago truncatula, MtSERF1 is strongly expressed in somatic embryos and its transcription depends on ethylene biosynthesis and perception (Mantiri et al., 2008). Over 50 genes encoding transcription factors, which take part in zygotic or somatic embryogenesis, have been identified and characterized in Arabidopsis. Among them there are genes involved in the initiation and maintenance of embryo development, such as LEC1 (Meinke, 1992), L1L (Kwong et al., 2003), LEC2 (Stone et al., 2001; Braybrook et al., 2006; Stone et al., 2008) and BBM (Boutilier et al., 2002). Their ectopic overexpression enables somatic tissues to acquire embryonic features inducing somatic embryogenesis on seedlings or tissues in culture, whereas their mutants show premature germination and non-embryonic characters in the embryos (Harada, 2001). Zinc finger proteins can act as DNA-binding transcription factors; however, some can bind to RNA. The CCCH zinc fingers recognize specific AU-rich sequences and interact with RNA (reviewed by Hall, 2005). For Arabidopsis and rice, the

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comprehensive computational analyses were done identifying 68 and 67 CCCH family genes, respectively (Wang et al., 2008). Expression studies indicated that CCCH proteins exhibit a variety of expression patterns, suggesting diverse functions. Compared with other gene families in rice and Arabidopsis thaliana, the CCCH gene family is one of the largest families in plants. In the present paper, we describe a new gene, CsSEF1 (Cucumis sativus Somatic Embryogenesis Zinc Finger 1), with expression sharply increased with the induction of somatic embryogenesis. The location of CsSEF1 transcripts in somatic embryos was determined by in situ hybridization. The possible effects of CsSEF1 overexpression and silencing of its close homologues were shown in transgenic Arabidopsis plants.

USA). The ‘driver’ cDNA was prepared from ECS before the induction of somatic embryogenesis and the ‘tester’ cDNA was prepared from tissue 2, 6, 12, 24 h, 3, 7, 14 d after the induction of embryogenesis. The subtracted PCR products generated by SSH were ligated to pBluescript SK(+) vector (Stratagene), transformed into Escherichia coli (XL1-Blue) (Stratagene) and plated on Luria-Bertani (LB) plates containing X-Gal/IPTG and ampicillin. The total 384 white colonies were selected for further analysis, preamplified, replicated in four 96well multiplates and arrayed on Hybond N nylon membranes (Amersham). To identify differentially expressed genes, reverse RNA hybridization analysis was performed with cDNA probes labeled using High Prime (Roche) and a32P dCTP (Amersham). The ‘tester’ and ‘driver’ cDNAs from subtracted and unsubtracted libraries were used in separate labeling reactions (four probes) and for hybridization with arrayed bacterial colonies according to Sambrook et al. (1989). Radioactive image analysis was performed using a Kodak K screen and Molecular Imager (BioRad).

Materials and methods Plant materials

cDNA library construction and screening

The embryogenic tissue was initiated from shoot tips of cucumber plants (Cucumis sativus L. var. Borszczagowski, denominated here ‘‘line B’’). The ECS was generated on MS medium (Murashige and Skoog, 1962) modified by Wro ´blewski et al. (1995). Induction of somatic embryos (SEs) from the ECS for the suppression subtractive hybridization (SSH) technique and for preparing a cDNA library have been described previously by Linkiewicz et al. (2004). The seeds of the Arabidopsis thaliana ecotype Landsberg erecta (Ler) and Columbia (Col-0) were obtained from the Nottingham Arabidopsis Stock Centre. The seeds of Arabidopsis T-DNA tagged lines with insertion in loci: At2g19810 (‘Arabidopsis thaliana zinc finger (CCCH-type) family protein gene’)-SALK_151571 and in At5g07500 (‘Arabidopsis thaliana PEI1; nucleic acid binding/transcription factor gene’)-SALK_108149 (Alonso et al., 2003) were provided by the Salk Institute Genomic Analysis Laboratory (La Jolla, CA, USA).

Poly(A)+ RNA (4 mg) extracted from 7-d-old cucumber somatic embryos were used to construct a library using the Stratagene ZAP-cDNAs Synthesis Kit and ZAP-cDNAs Gigapack III Gold Cloning Kit following the manufacturer’s instructions. The initial complexity of the library was 9.6  105 pfu. The 105 recombinants from the amplified library were used for screening plated at a density of 10,000 pfu/plate with competent XL1-Blue MRF’ cells. The phagemid screening was performed according to the manufacturer’s instructions using Porablot NCL membrane discs (Macherey–Nagel) and radiolabeled SSH fragment as a probe. The excision of the pBluescript candidate phagemids from Uni-ZAP XR vector was performed according to the manufacturer’s instructions (Stratagene). The candidate phagemids were amplified in SOLR E. coli strain and analyzed by EcoRI/XhoI restriction and sequencing.

RNA isolation from cucumber organs

DNA isolations and hybridization

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Total RNA was isolated using Trizol reagent (Invitrogen) according to the manufacturer’s instructions, from leaf, root, female flower, fruit, somatic embryos collected after 7 and 14 d post-SE induction and mix in equal proportion (w/w), mature zygotic embryos collected from 15-d-old fruits, and ECS. mRNA was isolated from the total RNA using Dynabeadss oligo-dT (Dynal) according to the manufacturer’s instructions. SSH library construction and screening The SSH technique (Diatchenko et al., 1999) was carried out using a PCR-Select cDNA Subtractive Kit according to the manufacturer’s protocol (Clontech,

Genomic DNA was extracted from cucumber leaves according to Michaels et al. (1994). For DNA hybridization blot, 5 mg of DNA (per sample) was digested and electrophoresed for 15 h (1.2 V/cm) in a 0.8% agarose gel, and then transferred on the Hybond N+ membrane (Amersham Pharmacia Biotech) according to Koetsier et al. (1993). Hybridization and washing were performed according to the membrane manufacturer’s protocol. Radioactive image analysis was performed using a Kodak K screen and Molecular Imager (BioRad). Full length cDNA of the gene CsSEF1 was used as a molecular probe for DNA hybridization. The PCR-amplified DNA fragments were purified using a QIAEX kit (Qiagen) and subsequently radiolabeled as described above.

ARTICLE IN PRESS CsSEF1 gene encoding putative CCCH-type zinc finger protein Real-time RT-PCR for CsSEF1 gene Real-time RT-PCR was performed with the LightCycler–RNA Amplification Kit SYBR Green I (Roche) according to manufacturer’s instructions (RT at 55 1C for 10 min, 95 for 30 s, 45 cycles of 95 1C for 0 s (slope 20 1C/s), 60 1C for 10 s, 72 1C for 30 s; product length 211 bp; primers: 50 -TCC ACC CAG ACC GAT ACC-30 and 50 -GGA GAC AAA GGT GGC GAG-30 ). The transcript level of CsSEF1 was estimated relative to cucumber 25S rRNA level (amplification conditions as above; product length 300 bp; primers: 50 -CCA GGT CAG GCG GGA CTA C-30 and 50 -CGC AAC GGG CTC TCT CAC C-30 ). The transcript levels are presented as values relative to ECS ( ¼ 1). Three independent experiments were performed. In situ hybridization Tissue fixation, embedding, sectioning and in situ hybridization with DIG-labeled sense and antisense RNA probes were performed as described by Long et al. (1996) with modifications described by Malinowski et al. (2004). Templates for the synthesis of RNA probes were prepared by cloning the CsSEF1 PCR fragment (1140 bp; primers: 50 -GCA CTT CCC ACC TCA TCT TCA-30 and 50 -ATT GTA ACT CAC TCA CTT GCC-30 ) into the pCRII-TOPO plasmid (Invitrogen). The protocol used was as follows: 30 cycles of 94 1C for 30 s, 60 1C for 30 s, 72 1C for 1 min. PCR was performed using a 20 mL reaction mixture that contained 20 ng of plasmid DNA, 200 mM each of dNTP, 500 nM of each primer, 1  Taq buffer with KCl, 1.25 mM MgCl2 and 1 U Taq DNA polymerase (Fermentas). The RNA probes were synthesized using SP6 and T7 RNA polymerases (Roche). Gene constructs and bacterial strains Transformation of Arabidopsis plants was performed using the Agrobacterium tumefaciens strain LBA4404 or EHA105 carrying pCAMBIA1380 vector (hygromycin resistance selection marker; Cambia, Canberra, Australia) modified by insertion of the 35S promoter at the EcoRI/ BamHI restriction site (A. Smigocki, Molecular Plant Pathology Laboratory, Agricultural Research Service, Beltsville, MD, USA) and appropriate gene fragments. For silencing, a 518 bp fragment of CsSEF1 cDNA clone, which was obtained from the SSH cDNA library, was introduced into the HindIII restriction site of pCAMBIA1380+35S. The T-DNA containing an antisense orientation of cDNA fragment was selected and designated as 35S::CsSEF1as. For overexpression, the CsSEF1 coding sequence (1071 bp) was amplified with specific primers containing HindIII and PstI restriction sites (underlined) added to the 50 end: 50 -CTA AGC TTA ATC AAT GCT CCT CAA TCC CA-30 and 50 -GAC TGC AGA TTG TAA CTC ACT CAC TTG CC-30 , restricted and ligated to pCambia 1380+35S vector. The PCR protocol used was as described above. This T-DNA was designated as 35S::CsSEF1s. Constructs for transformation were verified by sequencing of PCRamplified fragments and ligation sites.

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Growth conditions and transformation of Arabidopsis plants Germination of the seeds was synchronized by a treatment at 4 1C for 48 h in the dark. Plants were grown to the flowering stage in a growth chamber (Versatile Environmental Test Chamber MLR-35OH, Sanyo) at 22 1C with 10 h light (250 mE/m2 s) during the vegetative stage and 14 h light during generative growth (Wilson, 2000). Floral dip of plants was essentially as described by Clough and Bent (1998). The plants designated as T0 were grown to maturity and seeds were harvested. Seeds from floral-dipped plants (T1 generation) were surface sterilized and suspended in 0.1% sterile agarose and plated on selective medium (1/2 MS) (Murashige and Skoog, 1962) containing 1% sucrose, 0.8% agar, 15 mg/L hygromycin, 200 mg/L cefotaxime or 300 mg/L timentin. The growth conditions were as described above. After 3 weeks, resistant T1 seedlings were transferred to a fresh selection medium with 1.5% agar. After 1 week, some of these seedlings were put into the soil. The T2 seeds from each T1 plant were harvested and screened for the hygromycin resistance phenotype and phenotypic changes separately for each T1 progeny. Macroscopic observations of the hygromycin-resistant seedlings were made after 1, 3 and 4 weeks. Observations of the mature plants of T1 and T2 progeny were performed during and after flowering. Analysis of Arabidopsis transformants and mutants Genomic DNA from T1 and/or T2 transformants, mutants and non-transformed wild-type plants (control) was exTM tracted and PCR was performed by REDExtract-N-AMP Plant PCR Kit, according to the manufacturer’s protocol (Sigma). Antibiotic-resistant plants were screened by PCR for the presence of the hygromycin phosphotransferase gene (hpt) (primers: 50 -GGC GAG TAC TTC TAC ACA-30 and 50 -GCG AAG AAT CTC GTG CTT-30 , length product 887 bp long) and CsSEF1 cDNA sequence (50 -TCC ACC CAG ACC GAT ACC-30 and 50 -GGA GAC AAA GGT GGC GAG-30 , length product 211 bp long). The PCR protocol used was 30 cycles of 94 1C for 30 s, 62 1C (for hpt gene) or 60 1C (for CsSEF1 gene) for 30 s, 72 1C for 1 min (for hpt gene) or 30 s (for CsSEF1 gene). The insertions in T-DNA tagged lines (loci At2g19810 or At5g07500) and homozygous states were confirmed by three-primer-PCR with LBb1 (left border primer of pROK2; 50 -GCG TGG ACC GCT TGC TGC AAC T-30 ) and appropriate two genomic primers: 50 -TCG CCG TCG TGT TTG TTT CTT-30 and 50 -CAT TGC GAC TTG CAT CTT ACA TCA-30 for At2g19810; 50 -TTC CTT CAC GTA AAC GCT GCC30 and 50 -ACA CAA GTT CCC GGC GTT ACA-30 for At5g07500 locus. The protocol used was as follows: 34 cycles of 94 1C for 15 s, 60 1C for 30 s and 72 1C for 1 min.

Results Characterization of CsSEF1 sequence Differential screening of the SSH library involved hybridization of arrayed bacterial colonies with

ARTICLE IN PRESS 314 probes based on subtracted and unsubtracted cDNA isolated from tissues before and after induction of somatic embryogenesis. The application of the SSH strategy resulted in a normalized library and probes enriched in cDNAs characteristic for the stages preceding (probe cDNA only) and following the induction of somatic embryogenesis (probe and library). After the library screening, 117 clones were selected, of which the majority corresponded to transcripts induced during somatic embryogenesis (data not shown). The nucleotide sequences of the studied clones were compared with a protein sequence database using the algorithm blastx (Altschul et al., 1990). One of the clones, the 518 bp long CsSEF1, was chosen for further analyses. The sequence of this clone showed significant similarity to proteins possessing zinc fingers with atypical motif CCCH. The CsSEF1 fragment obtained from the SSH library was used to isolate seven independent clones from the conventional cDNA library. The sequence of the longest cDNA clone (1217 bp) was submitted to the EMBL database (accession number AJ870303). The sequence of the clone contains an 84 bp long 50 -UTR region, a 924 bp open reading frame and a 209 bp long 30 -UTR region. The putative protein coded by CsSEF1 consists of 307 amino acids. The amino acid sequence of CsSEF1 revealed the presence of three zinc finger motifs: the upper had the common pattern C-X5-H-X4-C-X3-H and two rarely found ones, middle C-X7-C-X5-C-X3-H and lower C-X5-CX4-C-X3-H (Figure 1). The protein coded by CsSEF1 showed the greatest similarity (using the algorithm blastp) to the Arabidopsis thaliana ‘zinc finger (CCCH-type) family protein’ (NP_194648, 56% within a 337 amino acid overlap, e-value 2e-66), Arabidopsis thaliana ‘zinc finger (CCCH-type) family protein’ (NP_179571, 56% similarity within a 345 amino acid overlap, e-value 1e65) as well as to ‘ATCTH; Arabidopsis thaliana Cys3His zinc finger protein’ (NP_180161, 60% similarity within a 318 amino acid overlap, e-value 2e62) and to the described Arabidopsis thaliana ‘PEI1; nucleic acid binding/ transcription factor’ (NP_196367, 70% similarity within a 127 amino acid overlap, e-value 2e38), which also contains CCCH motif. The multiple sequence alignment of CsSEF1 and four homologous proteins from Arabidopsis showed high similarity in the domain containing three zinc finger motifs (Figure 2A). The compared amino acid sequences from other species showed the highest conservation within the domain containing three zinc fingers (Figure 2B), while regions flanking this domain displayed high variability.

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Genomic DNA gel blot analysis for CsSEF1 gene In order to determine the number of copies of CsSEF1 in the cucumber genome, genomic DNA gel blot analysis was employed. The enzymes used in the analysis were selected with respect to the presence (for EcoRI, Eco32I) or absence (HindIII, XbaI) of appropriate restriction sites within the cDNA sequence. The molecular probe for the CsSEF1 clone was the entire cDNA sequence (1217 bp). As a result of hybridization for each restriction enzyme, a single strong signal was found, and in the case of enzymes recognizing a site within the studied sequence, a weaker second band was observed. These observations point to the existence of a single copy of CsSEF1 gene in the cucumber genome (Figure 3).

Analysis of CsSEF1 transcript accumulation The level of transcription of CsSEF1 was determined with the use of real-time semi-quantitative RT-PCR, and the results were standardized relative to control 25S rRNA gene from cucumber. An increased accumulation of transcripts in somatic embryos (SEs), leaves and female flowers, compared to ECS, was observed. The level of transcript accumulation in zygotic embryos and root, compared to ECS, was lower and similar, respectively (Figure 4). Similar results were obtained after RNA gel blot analysis (data not shown). In situ hybridization was carried out using cRNA sense and antisense probes specific for CsSEF1 mRNA to examine the spatial pattern of CsSEF1 expression in cucumber somatic embryos. Cucumber somatic embryos in different stages were transversely and longitudinally sectioned. Signals were observed in the globular, heart/torpedo and mature stages of embryos (Figure 5). The analysis demonstrated a high accumulation of transcripts in the putative apical domain of the late globular and early heart embryos (Figure 5A, B) and then in the cotyledon primordia of the heart embryo and older ones (Figure 5D, F). The transcript localization appears to be invariable during somatic embryo development. The transverse section also showed the presence of the CsSEF1 transcript in the procambium tissues, beginning from the heart stage onwards (Figure 5C). In the case of mature embryos with developed three cotyledon primordial, transcript accumulation was observed in every primordium (Figure 5F).

Transformation of Arabidopsis plants Arabidopsis plants were transformed with two gene expression cassettes. The T-DNA 35S::CsSEF1as

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Figure 1. Nucleotide and putative amino acid sequences of CsSEF1. Start codon ATG is marked by an ellipse. The restriction sites of enzymes used in DNA hybridization are underlined (besides HindIII and XbaI, which do not posses their sites in cDNA sequence); EcoRI: GAATTC; Eco32I: GATATC. Molecular probe for DNA gel blot was full length cDNA of CsSEF1. In putative protein sequence the characteristic zinc finger motifs are marked by double underline. Conservative cysteines and histidines are bold. First and last nucleotides of the sequence used to gene silencing are in boxes. The termination codon is marked with an asterisk. (A)n–poly(A) sequence.

for silencing contained the CsSEF1 fragment (518 bp) obtained from the SSH library, and the T-DNA 35S::CsSEF1s for overexpression carried a full length clone of CsSEF1 (1217 bp). The transformant seeds were plated on selective media. For both constructs the transformation efficiency was ca. 0.6%. For further analyses we selected 14 plants into which 35S::CsSEF1as had been introduced and 13 with 35S::CsSEF1s.

Molecular analysis of Arabidopsis transformants and mutants Stable integration of the transgenes was confirmed by PCR. Analysis with the use of primers specific for hpt gene demonstrated the presence of the expected product (910 bp) in all the transgenic

plants carrying the 35S::CsSEF1s or 35S::CsSEF1as cassettes (Figure 6A, B). The negative control was DNA isolated from non-transformed plants, whereas the positive control was plasmid DNA (pCAMBIA1380+35S) in equimolar concentration. PCR analysis was also performed to confirm the presence of CsSEF1 cDNA. In all the transformants into which 35S::CsSEF1s and 35S::CsSEF1as cassettes were introduced (except for two plants 5 and 9), a 211 bp long product was detected, indicating the integration of the T-DNA was correct for most of the plants (Figure 6C, D). These results were also confirmed by genome DNA gel blot hybridization using a molecular probe consisting of a fragment of the hygromycin phosphotransferase gene that was performed with selected transgenic lines (data not shown). The insertions in two T-DNA tagged Arabidopsis mutants (At2g19810 and At5g07500 loci) and

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Figure 2. (A) An alignment (ClustalW) of CsSEF1 (CAI30889.1) and the most closely related proteins and PEI1 from Arabidopsis thaliana (GenBank accession number-Arabidopsis locus ID: PEI1, NP_196367-At5g07500; NP_180161-At2g25900; NP_194648-At4g29190 and NP_179571-At2g19810). (B) An alignment of the domain containing three zinc finger motifs (ClustalW) of CsSEF1 and CAN83255 (Vitis vinifera), BAF04190 (Oryza sativa), ABK92961 (Populus trichocarpa), XP_001774473 (Physcomitrella patens subsp. patens), ABN08215 (Medicago truncatula) ABI30334 (Capsicum annuum). Identical, conserved and semi-conserved residues in all aligned sequences are indicated by asterisks (*), colons (:) and dots (.), respectively. Dashes (–) signify gaps.

homozygous states were confirmed by three-primerPCR, where two primers are complementary to the genomic sequences flanking the insertion site (giving

900 bp long product from the wild-type allele) and one primer is complementary to the T-DNA sequence (LBb1, 110 bp from the left border). The 400 bp long

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band that is present in our At5g07500 mutant analysis (700 bp long fragment; Figure 6E).

Phenotypic analysis of Arabidopsis transformants and mutants

Figure 3. DNA hybridization of cucumber genomic DNA. 5 mg of cucumber genomic DNA was digested with restriction enzyme indicated on each line. The blot was hybridized with the molecular probe which was full length cDNA of CsSEF1 gene. The estimated sizes for DNA bands are shown on right.

Figure 4. The expression of CsSEF1 gene in various organs. Transcript levels were analyzed by real-time RTPCR. Transcript levels are given as relative values to CES (the value of 1), after being normalized to the 25S ribosomal RNA levels. Data are shown as the means with variation bars (SE) from three independent reactions. ECS-embryogenic cell suspension, SEs–somatic embryos, ZEs–zygotic embryos. R–roots, Fl–female flowers, L–leaves.

PCR fragment in the case of insertion in At2g19810 locus and 500 bp long fragment in the case of At5g07500 locus confirmed the insertions of T-DNA in both mutants and also their homozygous state (Figure 6E). According to the Salk Institute Genomic Analysis Laboratory, the provider of the insertion mutants and methods to verify them, the LBb1 primer can sometimes produce an extra, non-specific

In the case of T1 hygromycin-resistant seedlings containing 35S::CsSEF1as cassette, three phenotypic classes could be distinguished. The first class consists of seedlings with a single cotyledon and reduced or normally developed root system (Figure 7A, C). The second phenotypic class contains seedlings that had developed two cotyledons and a reduced root system (Figure 7B). The third class consisted of seedlings with two cotyledons and a normally developed root system (Figure 7D). The percentage participation of individual classes was as follows: first class 62.5%, second 9.2% and third 28.3%. Only the seedlings from the third class were able to continue normal development. The mature plants obtained from these seedlings showed phenotypic alterations that mainly concerned the appearance of the siliques. In addition to siliques that looked the same as in the control plants (Figure 7G), siliques broader on the top (Figure 7H, I), shorter and flattened (Figure 7I, J), and showing other severe deformations (Figure 7N), could be distinguished. These changes occurred with varied intensity in all 14 analyzed plants. Seeds were properly developed only in normally appearing silique (Figure 7K). In the phenotypically altered silique, normally developed seeds were accompanied by seeds in which development was arrested at different stages (Figure 7L, M). In extreme cases, no seeds at all were observed (Figure 7N). In the transformation of wild-type Arabidopsis plants with a 35S::CsSEF1s cassette for overexpression and both types of mutants, no macroscopic changes at either the seedling stage or in the mature plants were observed. These plants developed normal cotyledons as well as rosettes, inflorescence shoots, siliques and produced seeds.

Discussion Zinc finger proteins belong to the most abundant group of proteins in eukaryotes. They have very different structures as well as functions, which include DNA or RNA recognition, RNA packaging, transcriptional activation, protein folding and assembly, and lipid binding. Laity et al. (2001) defined a zinc finger as any small, functional, independently folded domain that requires coordination of one or more zinc ions to stabilize its structure. Many

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Figure 5. In situ detection of CsSEF1 mRNA in cucumber somatic embryos. The signal was observed in the presumptive cotyledon primordia tissues of the globular stage of embryos (A), heart/torpedo (B) and torpedo stages of embryos (D, F) as well in procambium cells, from heart/torpedo stage (B) to mature stage (D). The transverse section shows also the presence of CsSEF1 transcript in the procambium tissues (C). The transcript accumulation was observed in primordia of embryos which generated three primordia (F). The scale bars represent 250 mm. Results come from representative experiments that were repeated at least five times with similar results. The in situ hybridization was carried out using cRNA antisense (A–D, F) and sense (E) probes specific for CsSEF1 mRNA. The arrows indicate cotyledon primordia (B, F) and procambium tissues (C, D).

proteins containing the classical CCHH zinc finger are transcription factors that function by recognition of specific DNA sequences, whereas those having the CCCH zinc finger motif often bind to RNA targets (Carballo et al. 1998; Tenlen et al. 2006; Pagano et al. 2007). CsSEF1 is one of the many genes with levels of expression that are increased with the induction of somatic embryogenesis. The full length of CsSEF1 cDNA codes a protein with three conserved zinc finger motifs, one with pattern CHCH (upper motif, different from the classical CCHH) and two with patterns CCCH (middle and lower motifs). These motifs are not the most typical; however, they are well conserved among proteins from different species. One of these plant proteins is PEI1, whose gene is the only CsSEF1 homologue from Arabidopsis with documented participation in embryogenesis. PEI1 is involved in the development of zygotic embryos from the globular stage to the heart stage

(Li and Thomas, 1998). Among other plant proteins with zinc finger motifs, only a few have been described that are involved in the development of embryos. One of these is RIE1, a novel RING-H2 finger protein with visible effects on seed development and constitutive expression in Arabidopsis. Mutation of the RIE1 gene was lethal under normal growing conditions, and mutant embryos were arrested from the globular to late torpedo stage (Xu and Li, 2003). There is also an interesting example of a protein that contains zinc finger motifs similar to those of the atypical middle and lower motifs in CsSEF1–HUA1 in Arabidopsis. This protein possesses six such motifs and belongs to a family of nine Arabidopsis genes containing multiple, tandem, CCCH-type zinc finger motifs (Li et al., 2001). HUA1 likely participates in a new regulatory mechanism governing flower development and acts only in the third and fourth whorls of the flower. No evidence exists for CCCH-type zinc

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Figure 6. PCR analysis of transgenic and mutant Arabidopsis plants. DNA was isolated from transgenic plants with the 35S::CsSEF1s (A, C) and with the 35S::CsSEF1as cassette (B, D). PCR amplification was done with primers for hpt gene (A, B) and for CsSEF1 gene (C, D). DNA was also isolated from plants of two mutant lines (E); one with the T-DNA insertion in At2g19810 locus (lines: 1–10) and second in At5g07500 (lines 12–21). DNA isolated from wild type plants (E, lines 11, 22). PCR amplification for confirmation of homozygous state of mutants was done with gene specific primer pair for At2g19810 locus (lines: 1–10) and gene specific primer pair for At5g07500 (lines 12–21) and third primer specific for T-DNA sequence, the same for both loci. CN-the negative control, DNA from non-transformed plants; CP-positive control, plasmid DNA (pCAMBIA1380+35S). M1, M2–DNA molecular markers, #SMO331 and #SMO311 (MBI Fermentas), respectively.

fingers being involved in DNA binding, but it was shown that this type of zinc finger binds RNA (namely AGAMOUS RNA fragments) or is associated with RNA metabolism (Cheng et al., 2003). The main criterion of CsSEF1 isolation was approximately nine-fold increase of the transcript accumulation after induction of somatic embryogenesis in cucumber ECS. Interestingly, levels of accumulation of CsSEF1 transcripts in ZEs (RNA isolated from mature stage ZEs) were at a lower level compared to ECS. This probably reflects the differences between somatic and zygotic embryogenesis, beginning with the fact that the acquisition of competence for embryogenesis is stimulated by different signals (Dodeman et al., 1997). The conditions in which the zygotic and somatic embryos develop are also quite different (embryo sac or in vitro culture). Therefore, the genes

participating in both somatic and zygotic embryogenesis can be activated or inhibited in different ways. The observed difference in transcript accumulation between SEs and ZEs could also result from the difference in developmental stages because most of cucumber somatic embryos took to RNA isolation represented late-heart and torpedo stages, whereas ZEs had developed cotyledons and came from developed seeds. Moreover, in the cucumber, in contrast to carrot or alfalfa, for example, there are significant differences in the anatomical structure between both kinds of embryos starting from late-heart stage (Tarkowska et al., 1994). An increased level of expression of the CsSEF1 gene was also found in the leaves and flowers. This may indicate that the gene also participates in later developmental stages after embryogenesis. In fact, there are only a few

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Figure 7. Phenotypes of 7-d-old seedlings of Arabidopsis thaliana T1 plants transformed with silencing construct (A-E). Seedling with single cotyledon and inhibited root growth (A), two cotyledons and reduced root growth (B), single cotyledon and reduced root growth (C), two cotyledons and normal root growth (D) can be distinguished and wild-type, control plant (E). Scale bar-3 mm. Siliques collected from wild-type, control plant (F) and T1 CsSEF1 antisense plants (G–N). Wild-type, control plant (F), normal (G), broader (H, I), short and flattened (J) and strongly deformed siliques (N) were observed. In opened siliques, seeds arrested in different stages were found (K–M). The arrows indicate several aborted seeds. Scale bars-1 mm.

ARTICLE IN PRESS CsSEF1 gene encoding putative CCCH-type zinc finger protein described genes in which activity is limited to only the process of embryogenesis. These include, for example, LEC1 in Arabidopsis thaliana (Meinke, 1992; Harada, 2001) and SERK in carrot (Schmidt et al., 1997). In the case of SERK, however, even though its expression in carrot is limited only to embryogenic tissues, the expression of its orthologues in other species (Arabidopsis thaliana and Medicago truncatula) was detected in additional tissues (Hecht et al., 2001; Nolan et al., 2003). The location of CsSEF1 transcripts in somatic embryos was limited to cotyledon/leaf primordia and procambium. However, Li and Thomas (1998) observed the expression of gene PEI1 from the globular to the late cotyledon stages equally distributed throughout the zygotic embryo. The expression patterns of genes PEI1 and CsSEF1 therefore do not overlap, and so their range of activities is most likely different. One of the many methods for studying the function of a gene is comparative analysis of plant phenotypes obtained after introducing gene constructs, resulting in the silencing of the gene or its overexpression. It is common knowledge that independent transformants carrying such constructs can differ considerably with respect to degree of silencing or overexpression, and this can depend on both the number of copies of the transgene, on its integration site and on many other factors (Filipecki and Malepszy, 2006). In order to carry out a preliminary analysis of gene functions, constructs for gene silencing and overexpression were introduced into the Arabidopsis genome. It was assumed that the level of similarity between cucumber gene and its homologues in Arabidopsis is sufficient for obtaining approximate information. This strategy also allowed the avoidance of the arduous cucumber transformation procedure, which is time consuming and induces a high degree of somaclonal variation. PCR analysis demonstrated the correctness of the integration of the transgene in most transgenic Arabidopsis plants obtained in this study. In our gene silencing experiment, the lack of cotyledon or deformation of siliques in Arabidopsis plants with 35S::CsSEF1as cassette indicates aberrations in the expression of gene/genes involved in the formation of appropriate primordia. Indeed, in cucumber CsSEF1 transcripts are present in cotyledon/leaf primordia and in female flower, where the fourth whorl is responsible for the gynoecium formation. In addition to the deformation of silique as such, an inhibition of the development of seeds inside them at varied stages was observed. Li and Thomas (1998), after the transformation of Arabidopsis plants with a coding sequence of PEI1 gene in

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antisense orientation, observed aberrations in the development of embryos, indicating a key role of this protein in the development of the embryos from the globular stage to the heart stage. Additionally, Li and Thomas (1998) observed morphological changes in the seedlings. They obtained seedlings with one, two and multiple leaf primordia, and some of these developed regular leaves. The authors suggest that PEI1 may also be required for the normal development of cotyledons that are formed already at the heart stage as well as being involved in the proper formation of shoot apical meristem. Silencing of a gene with the use of an antisense is frequently problematic. It is difficult to show whether only one target gene is silenced. This situation resembles the recently discussed cases of a non-specific effect (‘off-target effect’) that can be induced by siRNA (Jackson and Linsley, 2004). Based on a comparison between the nucleotide sequence of CsSEF1 and the Arabidopsis genome, the introduction of 35S::CsSEF1as cassette could result in the silencing of the most similar genes: At2g25900 and/or At2g19810. Caution in the interpretation of phenotypes obtained as a result of RNAi experiments postulated by Jackson and Linsley (2004) concerns, above all, multigene families. Li and Thomas (1998) achieved silencing by introducing the entire PEI1-coding sequence in the antisense orientation and observed arrested development of Arabidopsis thaliana embryos at the globular stage. In this case, the silencing of other homologues that could have yielded the observed effect cannot be excluded. Such an idea is confirmed by our preliminary macroscopic observations of the homozygous insertion mutant pei1 (SALK_108149), in which insertion of T-DNA following codon 57 leads to the formation of a 58 amino acid oligopeptide with no zinc finger motifs and in which no developmental disorders were detected (data not shown). The seeds obtained from mentioned mutant germinated, giving rise to normal seedlings. It seems that the lack of functional protein PEI1 in the mutant has no lethal effect on the development or that the function of the gene has been taken over by its homologue. The silenced phenotype described by Li and Thomas (1998) could have been enhanced by the non-specific silencing of other close homologues of the PEI1 gene. In the case of our overexpression experiment, the lack of phenotypic changes is difficult to explain. It might be that cucumber protein lacks the specificity for interaction with other proteins or nucleic acids from Arabidopsis. The putative CsSEF1 protein has motifs that are characteristic

ARTICLE IN PRESS 322 for RNA-binding proteins and it may thus be involved in the post-transcriptional regulation of gene expression influencing mRNA stability. Such specific interactions with sequences within 30 -UTR of target mRNA were shown for mammalian CCCH zinc finger proteins TTP (tristetraprolin) and Caenorhabditis elegans MEX-5 (Brewer et al., 2004; Pagano et al., 2007). The potential role of CsSEF1 in binding the specific mRNA regions requires confirmation by experimental approaches in the future.

Acknowledgments This work was supported by Polish Ministry of Science and Higher Education (grant no. PBZ/KBN/ 029/P06/2000).

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