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Isolation of two somatic embryogenesis-related genes from orchardgrass (Dactylis glomerata)
Plant Science 162 (2002) 301– 307 www.elsevier.com/locate/plantsci
Isolation of two somatic embryogenesis-related genes from orchardgrass (Dactylis glomerata) Krassimira S. Alexandrova, B.V. Conger * Department of Plant and Soil Sciences, Uni6ersity of Tennessee, Knox6ille, TN 37901 -1071, USA Received 31 July 2001; received in revised form 26 October 2001; accepted 28 October 2001
Abstract Orchardgrass (Dactylis glomerata L.) leaf segments have a high capacity for direct embryogenesis from mesophyll cells and indirect embryogenesis through callus. Total RNA extracted from leaf cultures of embryogenic and nonembryogenic genotypes were used to generate two differentially expressed cDNA fragments. An embryogenic leaf culture cDNA library was screened with these fragments and two somatic embryogenesis-related genes designated DGE1 and DGE2 (for Dactylis glomerata embryogenesis) were identified. RNA blot analysis showed that the DGEs were expressed in embryogenic but not in nonembryogenic leaf cultures. DGE1 transcripts were detected in leaf cultures induced for direct and indirect embryogenesis, while DGE2 was found only in leaf cultures induced for direct embryogenesis. A 90 aa segment of DGE1 showed 81% identity with the WRKY DNA-binding protein 21 from Arabidopsis thaliana and contained the WRKY DNA-binding domain. The entire DGE2 sequence had no significant homology with any sequences contained in the Gene Bank. Both genes encode proteins with putative nuclear localization sequences. Collectively, our data indicate that DGE1 and DGE2 are somatic embryogenesis-related genes with possible nuclear regulatory functions. © 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Dactylis glomerata; Orchardgrass; Differential display; Somatic embryogenesis
1. Introduction Somatic embryogenesis in Poaceae species is a process of producing embryos from immature, undifferentiated somatic cells [1,2]. The initiation of embryogenic cells requires culturing the appropriate explant on induction medium containing plant growth regulators (PGRs) such as auxins [3]. Gene expression during somatic embryogenesis is believed to follow the same developmental pathway as that for zygotic embryogenesis. All seed producing plants possess somatic cells with the necessary genetic information for embryogenesis, but not all of these cells can form embryos. Therefore, somatic embryo formation depends on the ability of a plant cell to enter a new developmental program [4]. ‘Embryogen-P’ orchardgrass has a high capacity for production of somatic embryos from leaf cultures, * Corresponding author. Tel.: + 1-865-974-8833; fax: +1-865-9747997. E-mail addresses: [email protected] (K.S. Alexandrova), [email protected] (B.V. Conger).
which makes it a model system for studying fundamental processes of embryogenesis in Poaceae [5]. An additional advantage of this system includes a gradient of embryogenic responses within the basal 30 mm of the youngest/innermost leaves. The most basal portion gives rise to both embryogenic callus and somatic embryos, with direct somatic embryo formation only gradually replacing the embryogenic callus formation in the more distal portions. Direct somatic embryogenesis decreases in frequency above 20 mm from the base until eventually no response is observed. Apparently, the gradient of embryogenic response coincides with the gradient of leaf tissue differentiation. The younger basal tissue with more actively dividing cells is prone to callus formation in addition to direct embryo formation, while the cells in more differentiated distal regions, where the rate of cytokinesis is reduced, appear to produce only direct embryos. Although, there has been significant progress toward identification of genes involved in the induction of somatic embryogenesis [6–9], our understanding of the
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molecular events during this process is still limited. We used embryogenic and nonembryogenic orchardgrass genotypes to study gene expression during direct and indirect somatic embryogenesis. This paper describes the isolation and characterization of two genes expressed in embryogenic, but not in nonembryogenic leaf cultures.
2. Materials and methods
2.1. Leaf cultures The highly embryogenic genotype Embryogen-P [5], and a nonembryogenic genotype, Nonembryogen, were maintained under greenhouse conditions. Both genotypes originate from the cultivar Potomac. The basal 30 mm of the two innermost leaves of tillers were split longitudinally along the midvein and surface sterilized for 2 min in a 2.62% NaOCl solution containing 0.1% w/v Triton-X. The leaf halves were then washed in three changes of sterile distilled water. Each leaf half was cut transversely into six equal segments. The segments from one half of each leaf were serially explanted onto Petri plates containing Schenk and Hildebrandt [10] medium without growth regulators (SH0). Corresponding segments from the remaining half were plated onto the same medium but amended with 30 mM of the synthetic auxin 3,6-dichloro-2methoxybenzoic acid, also known as dicamba (SH30). The pH of SH0 and SH30 media was adjusted to 5.8 with 1 M NaOH prior to autoclaving. The leaf segments were then incubated in the dark at 22 °C for 4, 11, 17 or 28 days. The basal three segments of each treatment were collected separately from the distal three segments and then stored at −80 °C until total RNA isolation.
2.2. RNA isolation and mRNA differential display Total RNA was isolated using an RNeasy Kit (Qiagen, Valencia, CA), according to the manufacturer’s instructions. The RNAimage Kit (GenHunter, Nashville, TN) was used for RT-PCR differential display [11]. Three one-base-anchored oligo-dT primers, H-T11G, H-T11A, and H-T11C in combination with eight arbitrary primers, H-AP49 through H-AP56 (Table 1) were used for PCR amplification according to the manufacturer’s protocol. This method was first devised for human cells with the assumption that every cell expresses 15 000 genes [12]. To display all genes expressed, the method requires at least 25 arbitrary primers to achieve a level of confidence of about 0.95. Assuming that orchardgrass cells express the same number of genes, we can speculate that our study covered one third of them. The amplified cDNA frag-
ments labeled with 33P-dCTP were separated in a denaturing 6% polyacrylamide gel. The fragments were then visualized on a Kodak Biomax MS film by autoradiography. RT-PCR differential display was repeated three times for the reactions that produced differentially expressed cDNAs. Only bands that appeared repeatedly in the three independent reactions were selected, extracted from the gel and reamplified in a PCR reaction, as described by the kit manufacturer. The reamplified cDNA from each band was cloned in a pCR2.1 vector. Plasmids containing a DNA fragment were isolated from randomly selected bacterial colonies and then sequenced.
2.3. cDNA library construction and screening Basal leaf segments cultured on SH30 for 4 and 11 days, and basal and distal leaf segments cultured on SH30 for 25 days were used for mRNA isolation. A unidirectional cDNA library was constructed with the pooled mRNA in the Uni-ZAP XR vector (Stratagene, La Jolla, CA). The cDNAs were size fractionated prior to vector insertion. Fraction no. 1 contained the largest cDNAs and was used for screening. A minimum of twenty 150-mm agar plates with 50 000 pfu/plate was used for each screening. Plaque lifts were done with 132 mm discs of Hybond-N (Amersham, Piscataway, NJ). Probes were prepared by PCR amplification of a cDNA fragment under the same conditions as described for differential display, only with different nucleotide concentrations. Twenty micromolar each of dATP, dGTP, and dTTP, 10 mM of dCTP, and 5 ml of 10 mCi/ml [a-32P]dCTP were used in a 50 ml reaction. Five microliter of 1:100 dilution of the first-round PCR sample was used as a template. Membranes were hybridized in HYB-9 hybridization solution (Gentra, Minneapolis, MN) at 65 °C and then washed with a medium stringency wash solution (0.2× HYB-9) for approximately 2 h at 65 °C. Plaques that produced a hybridization
Table 1 Three anchored and eight arbitrary primers used for differential display of orchardgrass cultures Primers
Primer sequence (5%–3%)
H-T11G H-T11A H-T11C H-AP49 H-AP50 H-AP51 H-AP52 H-AP53 H-AP54 H-AP55 H-AP56
AAGCTTTTTTTTTTTG AAGCTTTTTTTTTTTA AAGCTTTTTTTTTTTC AAGCTTTAGTCCA AAGCTTTGAGACT AAGCTTCGAAATG AAGCTTGACCTTT AAGCTTCCTCTAT AAGCTTTTGAGGT AAGCTTACGTTAG AAGCTTATGAAGG
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signal were selected, and purified. Phagemid excision from the Uni-ZAP XR vector was done in vivo with ExAssist helper phage (Stratagene, La Jolla, CA) according to the manufacturer’s protocol.
2.4. RNA blot analysis Ten mg of total RNA from each RNA preparation were loaded onto a 1% agarose gel with formaldehyde and then blotted onto a Hybond-N membrane (Amersham, Piscataway, NJ). The membranes were pre-hybridized at 65 °C for 2 h in a solution containing 5% SDS, 0.33 M of sodium phosphate, 0.1 M of EDTA and 100 mg/ml salmon sperm DNA. Probes were synthesized with the RadPrime DNA labeling system (Gibco BRL, Grand Island, NY). An actin probe was made from a 500 bp actin fragment after EcoRI digestion of an Act5 clone. This clone contained the first actin exon from Lotus japonicus L., inserted in the pCR2.1-TOPO vector (Invitrogen, Carlsbad, CA). The membranes were washed at 65 °C with 2× SSC + 1% SDS for 1 h and with 0.1 × SSC + 0.5% SDS for an additional 1 h. They were then exposed to Kodak Biomax MS films for 10 – 14 days.
2.5. DNA blot analysis Genomic DNA was isolated from leaves using the Puregene DNA isolation kit (Gentra, Minneapolis, MN). Ten microgram of DNA was digested with HindIII, XhoI, BglII or BamHI, separated on a 0.8% agarose gel and blotted onto Hybond-N membranes (Amersham, Piscataway, NJ). The blots were pre-hybridized at 65 °C in HYB-9 hybridization solution (Gentra, Minneapolis, MN). Hybridization was performed with random primed 32P-labeled probes at 65 °C overnight. The membranes were then washed with a medium stringency 0.2× HYB-9 solution at 65 °C.
2.6. Sequencing and analysis DNA sequencing was performed with the ABI Prism Dye Terminator Cycle Sequencing reaction kit on an ABI 373 DNA sequencer (Perkin– Elmer, Foster City, CA). The initial sequence data text files were edited following comparison with the same data displayed in four-color electrophoregrams before they were analyzed further. Sequence data were analyzed using the Baylor College of Medicine (BCM) Search Launcher and the Expert Protein Analysis System (ExPASy) proteomics server of the Swiss Institute of Bioinformatics. Database searches were conducted with the basic local alignment search tool (BLAST) of the National Center for Biotechnology Information (NCBI).
Fig. 1. RT-PCR differential display of distal leaf segments after 28 days of culture. RT was performed with primer H-T11A (A), and H-T11C (B). PCR was carried out with the same anchored primer in combination with H-AP52 (A) and H-AP55 (B). Lane 1-Nonembryogen on SH0, lane 2-Nonembryogen on SH30, lane 3-Embryogen-P on SH0 and lane 4-Embryogen-P on SH30. Isolated bands marked with an arrow.
3. Results Because of the much lower response of distal leaf segments (a high proportion of the cells have already undergone differentiation), total RNA was isolated from these segments of Nonembryogen and Embryogen-P after 28 days culture on SH0 or SH30 medium. This RNA was used for RT-PCR differential display and their cDNA patterns were compared. Bands that appeared only in Embryogen-P cultured on SH30 medium were selected in order to avoid the cDNAs whose expression was induced by dicamba, but that was not related to somatic embryogenesis. Thus, eight cDNA bands were isolated from 24 reactions, but only two were used for further analyses (Fig. 1). Fragments D28.A52 and D28.C55b were 332 and 252 nucleotides in length, respectively, and represented the 3% end of two distinct mRNAs (data not shown). They showed
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no significant similarity with sequences in the NCBI GeneBank database. To determine the complete sequences of the differentially expressed cDNAs, fragments D28.A52 and D28.C55b were used as probes to screen a cDNA library of basal and distal embryogenic leaf cultures. Three independent clones were isolated from the cDNA library with the cDNA fragment D28.A52. The largest clone, DGE1 [accession no. AY011121] was 1733 nucleotides. Another clone, was only 5 bp shorter than DGE1 at the 5% end and was therefore named DGE1a. The third clone isolated with the same fragment was 1001 bp and was identical to the 3% end of DGE1. It was designated as DGE1b. The DGE1 ORF translated into a 386 aa polypeptide (Fig. 2). A BLAST homology search showed that a 90 aa region of DGE1 had 81% identity with the WRKY DNA-binding protein 21 from Arabidopsis thaliana (Fig. 3). The polypeptide sequence in the region of similarity contained an entire WRKY domain that is characteristic of the WRKY superfamily of plant transcription factors [13]. DGE1 contained glutamine-rich (180– 197) and serine-rich (222– 265) regions (Fig. 2), which are also characteristic of the WRKY proteins. Additionally, there was 87.9% identity between 1000 nucleotides at the 3% end of DGE1, part of which encoded the WRKY domain, and a clone
Fig. 2. DGE1 cDNA sequence and translation of the longest ORF. A stop codon is marked with a ‘*’. Glutamine-rich (180 –197) and serine-rich (222 – 265) regions are highlighted. The nuclear localization signal is underlined.
Fig. 3. Comparison of a segment from DGE1 containing the WRKY domain with other WRKY proteins. The alignment includes the amino acid sequence of WRKY protein 21 from Arabidopsis thaliana [accession no. AF272747], WRKY3 from A6ena sati6a L., oat [accession no. AF140553], WRKY transcription factor Nt-SubD48 from Nicotiana tabacum, tobacco [accession no. AB041520], and WRKY3 from Petroselinum crispum L, parsley [accession no. U56834]. Location of identical amino acids in at least four organisms is highlighted.
from a 4–45 days after pollination spike EST library of barley (Hordeum 6ulgare L.) [accession no. BE194160]. Furthermore, a 78.7% identity was found over 361 nucleotides starting at position 140 with a cDNA from rice (Oryza sati6a L.) etiolated leaf sheath [accession no. AI978441]. Analyses of the DGE1 polypeptide suggested that it was most likely a soluble protein with a theoretical pI of 9.97 and a relative molecular mass of 41 700. A nuclear targeting sequence was predicted by the PSORT/ExPaSy at position 297–300 (Fig. 2). DGE2 [accession no. AY011122] was isolated from the cDNA library with fragment D28.C55b. It had 1378 nucleotides with an ORF that encoded a 354 aa polypeptide (Fig. 4). Searches of the GenBank/NCBI databases showed that a 423 nucleotide segment of the clone, starting at residue 182, was 47.8% identical to a sorghum (Sorghum bicolor L.) pathogen induced PI1 clone [accession no. BE600715]. Furthermore, a 79.5% identity with a clone from a wheat (Triticum aesti6um L.) pre-anthesis spike cDNA library [accession no. BE500740] was exhibited over 410 nucleotides starting at position 624. DGE2 showed no significant similarity with DGE1. The primary sequence analyses of DGE2 showed that it was likely a soluble protein with a pI of 10.1 and a molecular mass of 41 500. The polypeptide had a high, approximately 20%, arginine content. Three nuclear targeting sequences were predicted by the PSORT/ExPaSy Molecular Biology Server. Two are K and R rich regions and the third is known as the Robbins and Dingwall consensus [14], which is defined by two basic residues, a ten residue spacer, and another basic region consisting of at least three basic residues out of five. The differential expression of the isolated clones was confirmed by RNA blot analysis (Fig. 5). Low expression levels of DGE1 were observed in basal leaf segments at 4, 11, 17 and 28 days, and also in distal leaf segments at 17 and 28 days of culture on SH30 medium. DGE1 produced no hybridization with DNA
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from leaf segments cultured on SH0 medium. The hybridization signal produced by DGE2 was limited to distal leaf segments cultured on SH30 for 28 days. Genomic DNA separately digested with HindIII, XhoI, BglII or BamHI was subjected to DNA hybridization with the cDNAs for DGE1 or DGE2 as a probe. Two or three hybridizing bands were found in each lane under a medium-stringency condition (Fig. 6). This is an indication that there are probably two DGE1-related and two DGE2-related genes in Embryogen-P.
4. Discussion Embryogenic orchardgrass leaf cells are located primarily in the leaf mesophyll subjacent to both the adaxial and abaxial leaf surfaces [15]. The initial divisions are observed as early as 4 days after culture initiation. Approximately 70– 80% of the initial divisions are periclinal to the leaf surface [16]. Divisions of the daughter cells occur 6– 8 days after initiation of cultures. The culture time necessary for initiation of the first divisions corresponds to the earliest time of detection of somatic embryogenesis receptor-like kinase (SERK) transcripts [17]. SERK was proposed to be a marker for cells competent to form embryos [8]. In the present study, a RT-PCR differential display technique [11] was used to identify genes that were
Fig. 4. DGE2 cDNA sequence and translation of the longest ORF. A stop codon is marked with a ‘*’. Predicted nuclear targeting K and R rich sequences are underlined and a sequence matching the Robbins and Dingwall consensus for a bipartite nuclear localization signal is marked with a double line.
Fig. 5. RNA blot analyses of DGE1 and DGE2. An actin clone Act5 from Lotus japonicus was used as a control. Ten milligram total RNA from Embryogen-P was loaded in each lane. RNA was isolated from basal and distal leaf segments cultured for 4, 11, 17, or 28 days on SH0 or SH30 medium. Embryogenic capacity was measured as a number of embryos developed into plantlets after induction of leaf segments on SH30 medium for 4, 11, 17 or 28 days, followed by culture on SH0 for 30 days.
expressed during direct or indirect embryogenesis. This method is advantageous to the numerous variations of subtractive hybridization procedures because the expression of more than two sets of samples can be monitored simultaneously. Four sets of samples were compared that represented three different controls and one sample in which somatic embryogenesis occurred. The expression patterns were compared for leaf segments from Embryogen-P and Nonembryogen cultured on SH0 or SH30. Presumably, Nonembryogen leaf segments responded to dicamba in the same way as Embryogen-P except that embryogenesis was not induced. Therefore, Nonembryogen was used to eliminate the genes induced by dicamba, but not involved in the process of somatic embryogenesis. In total, 24 pairs of primer combinations were used and eight bands were identified which were induced by dicamba in embryogenic leaf cultures. The cDNA from two of the bands was used to prepare PCR probes for library screening. Thus, two unique clones, DGE1 and DGE2, were isolated from the cDNA library, that corresponded to mRNAs induced during somatic embryogenesis. The DGEs were differentially expressed in embryogenic tissues. Low levels of DGE1 transcripts were detected in embryogenic basal segments over the entire course of study and distal leaf segments starting at 17 days. This suggested that DGE1 might play a role in
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indirect and direct embryogenesis, or alternatively is a reflection of the basal leaf cell forming direct embryos. Distal leaf segments after 17 days culture on SH30 medium, produced only a few embryos, but more leaf cells might have been competent for embryogenesis at that time. The expression of DGE1 in these segments implied that it might have a role in the induction of embryogenesis. The DGE2 probe only hybridized to RNA from embryogenic distal leaf segments cultured for 28 days on SH30. This suggested that DGE2 may not be expressed in embryogenic basal leaf segments and therefore, indirect embryogenesis in basal leaf and direct embryogenesis in the more distal leaf cells might follow different pathways. Neither DGE1 nor DGE2 transcripts were detected in RNA from nonembryogenic leaf segments cultured on SH0 medium. DGE1 encodes a 386 aa polypeptide predicted to be a soluble protein with a pI of 9.97 and to have a nuclear targeting sequence. A high similarity was found between the C-termini of DGE1 and WRKY21 from Arabidopsis that contained a WRKY domain and a nuclear targeting sequence. WRKY21 is a member of the WRKY superfamily of sequence-specific DNAbinding proteins that have been found only in plants. This is a family of transcriptional regulators involved in senescence and trichome development [13]. Furthermore, they play a key role in a signal transduction pathway that leads from elicitor perception to the activation of pathogenesis-related genes from class 1 [18].
These facts suggest that DGE1 resides primarily in the nucleus and might have a transcriptional regulatory function. In addition, the accumulation of three PR proteins was correlated with somatic embryogenesis [19]. Therefore, the partial similarity of DGE1 to a gene involved in response to pathogens suggested that similar proteins are involved in the induction of somatic embryogenesis and PR proteins. The putative DGE2 protein shared certain features with that of DGE1. It was a hydrophilic protein with a pI of 10.1 that contained putative nuclear targeting sequences. However, no sequence homology was found between DGE1 and DGE2. The sequence similarity of one part of DGE2 with a pathogen induced clone PI1 from Sorghum bicolor and another part with a clone from a wheat pre-anthesis spike cDNA library suggested that DGE2 might share domains with other proteins, but their role remains unknown. In conclusion, two genes, DGE1 and DGE2 were identified to be associated with somatic embryo formation in orchardgrass. These two genes are likely to encode nuclear proteins with possible regulatory functions and may be responsible for some of the mechanisms essential for the formation of embryogenic plant cells, especially in Poaceae. Acknowledgements The authors thank Dr Neil Quigley from the Molecular Biology Resource Facility at UTK for the DNA sequencing, and Drs Albrecht von Arnim and Beth Mullin from the Department of Botany at UTK for their support and assistance in the preparation of the manuscript. We also thank Ledare Habera for professional guidance throughout the project. References
Fig. 6. Genomic DNA blot analysis of DGE1 and DGE2. Ten microgram DNA was digested with HindIII (H), XhoI (X), BglII (Bg) or BamHI (Ba). After blotting the DNA was probed with cDNAs from DGE1 or DGE2. Both DGE1 and DGE2 clones lack restriction sites for the enzymes used. Sizes of DNA markers are indicated on the left.
[1] W. Wernicke, R. Brettell, Somatic embryogenesis from Sorghum bicolor leaves, Nature 287 (1980) 138 – 139. [2] B.V. Conger, G.E. Hanning, D.J. Gray, J.K. McDaniel, Direct embryogenesis from mesophyll cells of orchardgrass, Science 221 (1983) 850 – 851. [3] F. Lo Schiavo, Early events in embryogenesis, in: Y.P.S. Bajaj (Ed.), Biotechnology in Agriculture and Forestry, vol. 30, Springer, Berlin, 1995, pp. 20 – 29. [4] Y. Henry, P. Vain, J. De Buyser, Genetic analysis of in vitro plant tissue culture responses and regeneration capacities, Euphytica 79 (1994) 45 – 58. [5] B.V. Conger, G.E. Hanning, Registration of Embryogen-P orchardgrass germplasm with a high capacity for somatic embryogenesis from in vitro cultures, Crop Sci. 31 (1991) 855. [6] G.R. Heck, S.E. Perry, K.W. Nichols, D.E. Fernandez, AGL15, a MADS domain protein expressed in developing embryos, Plant Cell 7 (1995) 1271 – 1282. [7] M.K. Filipecki, H. Sommer, S. Malepszy, The MADS-box gene CUS1 is expressed during cucumber somatic embryogenesis, Plant Sci. 125 (1997) 63 – 74.
K.S. Alexandro6a, B.V. Conger / Plant Science 162 (2002) 301–307 [8] E. Schmidt, F. Guzzo, M.A.J. Toonen, S.C. de Vries, A leucine rich repeat containing receptor-like kinase marks somatic plant cells competent to form embryos, Development 124 (1997) 2049 – 2062. [9] T. Lotan, M. Ohto, K. Matsudaira, Arabidopsis LEAFY COTYLEDON1 is sufficient to induce embryo development in vegetative cells, Cell 93 (1998) 1195 –1205. [10] R.U. Schenk, A.C. Hildebrandt, Medium and techniques for induction and growth of monocotyledonous and dicotyledonous plant cell cultures, Can. J. Bot. 50 (1972) 199 –204. [11] P. Liang, L. Averboukh, K. Keyomarsi, R. Sager, A.B. Pardee, Differential display and cloning of messenger RNAs from human breast cancer versus mammary epithelial cells, Cancer Res. 52 (1992) 6966 – 6968. [12] D. Bauer, H. Muller, J. Reich, H. Riedel, V. Ahrenkiel, P. Warthoe, M. Strauss, Identification of differentially expressed mRNA species by an improved display technique (DDRT-PCR), Nucleic Acid Res. 21 (1993) 4272 –4280. [13] T. Eulgem, P.J. Rushton, S.E. Robarzek, I.E. Somssich, The WRKY superfamily of plant transcription factors, Trends Plant Sci. 5 (2000) 199 – 206. [14] J. Robbins, S.M. Dilworth, R.A. Laskey, C. Dingwall, Two
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interdependent basic domains in nucleoplasmin nuclear targeting sequence: identification of a class of bipartite nuclear targeting sequence, Cell 64 (1991) 615 – 623. R.N. Trigiano, D.J. Gray, B.V. Conger, J.K. McDaniel, Origin of direct somatic embryos from cultured leaf segments of Dactylis glomerata, Bot. Gaz. 150 (1989) 72 – 77. A. Vasilenko, J.K. McDaniel, B.V. Conger, Ultrastructural analyses of somatic embryo initiation, development and polarity establishment from mesophyll cells of Dactylis glomerata, In Vitro Cell. Dev. Biol.-Plant 36 (2000) 51 – 56. M. Somleva, E.D.L. Schmidt, S.C. de Vries, Embryogenic cells in Dactylis glomerata L. (Poaceae) explants identified by cell tracking and by SERK expression, Plant Cell Rep. 19 (2000) 718 – 726. T. Eulgem, P.J. Rushton, E. Schmelzer, K. Hahlbrock, I.E. Somssich, Early nuclear events in plant defense signaling: rapid gene activation by WRKY transcription factors, EMBO J. 18 (1999) 4689 – 4699. S. Helleboid, T. Hendriks, G. Bawn, D. Inze, J. Vasseur, J.-L. Hilbert, Three major somatic embryogenesis related proteins in Cichorium identified as PR proteins, J. Exp. Bot. 51 (2000) 1189 – 1200.
Isolation of two somatic embryogenesis-related genes from orchardgrass (Dactylis glomerata) Krassimira S. Alexandrova, B.V. Conger * Department of Plant and Soil Sciences, Uni6ersity of Tennessee, Knox6ille, TN 37901 -1071, USA Received 31 July 2001; received in revised form 26 October 2001; accepted 28 October 2001
Abstract Orchardgrass (Dactylis glomerata L.) leaf segments have a high capacity for direct embryogenesis from mesophyll cells and indirect embryogenesis through callus. Total RNA extracted from leaf cultures of embryogenic and nonembryogenic genotypes were used to generate two differentially expressed cDNA fragments. An embryogenic leaf culture cDNA library was screened with these fragments and two somatic embryogenesis-related genes designated DGE1 and DGE2 (for Dactylis glomerata embryogenesis) were identified. RNA blot analysis showed that the DGEs were expressed in embryogenic but not in nonembryogenic leaf cultures. DGE1 transcripts were detected in leaf cultures induced for direct and indirect embryogenesis, while DGE2 was found only in leaf cultures induced for direct embryogenesis. A 90 aa segment of DGE1 showed 81% identity with the WRKY DNA-binding protein 21 from Arabidopsis thaliana and contained the WRKY DNA-binding domain. The entire DGE2 sequence had no significant homology with any sequences contained in the Gene Bank. Both genes encode proteins with putative nuclear localization sequences. Collectively, our data indicate that DGE1 and DGE2 are somatic embryogenesis-related genes with possible nuclear regulatory functions. © 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Dactylis glomerata; Orchardgrass; Differential display; Somatic embryogenesis
1. Introduction Somatic embryogenesis in Poaceae species is a process of producing embryos from immature, undifferentiated somatic cells [1,2]. The initiation of embryogenic cells requires culturing the appropriate explant on induction medium containing plant growth regulators (PGRs) such as auxins [3]. Gene expression during somatic embryogenesis is believed to follow the same developmental pathway as that for zygotic embryogenesis. All seed producing plants possess somatic cells with the necessary genetic information for embryogenesis, but not all of these cells can form embryos. Therefore, somatic embryo formation depends on the ability of a plant cell to enter a new developmental program [4]. ‘Embryogen-P’ orchardgrass has a high capacity for production of somatic embryos from leaf cultures, * Corresponding author. Tel.: + 1-865-974-8833; fax: +1-865-9747997. E-mail addresses: [email protected] (K.S. Alexandrova), [email protected] (B.V. Conger).
which makes it a model system for studying fundamental processes of embryogenesis in Poaceae [5]. An additional advantage of this system includes a gradient of embryogenic responses within the basal 30 mm of the youngest/innermost leaves. The most basal portion gives rise to both embryogenic callus and somatic embryos, with direct somatic embryo formation only gradually replacing the embryogenic callus formation in the more distal portions. Direct somatic embryogenesis decreases in frequency above 20 mm from the base until eventually no response is observed. Apparently, the gradient of embryogenic response coincides with the gradient of leaf tissue differentiation. The younger basal tissue with more actively dividing cells is prone to callus formation in addition to direct embryo formation, while the cells in more differentiated distal regions, where the rate of cytokinesis is reduced, appear to produce only direct embryos. Although, there has been significant progress toward identification of genes involved in the induction of somatic embryogenesis [6–9], our understanding of the
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molecular events during this process is still limited. We used embryogenic and nonembryogenic orchardgrass genotypes to study gene expression during direct and indirect somatic embryogenesis. This paper describes the isolation and characterization of two genes expressed in embryogenic, but not in nonembryogenic leaf cultures.
2. Materials and methods
2.1. Leaf cultures The highly embryogenic genotype Embryogen-P [5], and a nonembryogenic genotype, Nonembryogen, were maintained under greenhouse conditions. Both genotypes originate from the cultivar Potomac. The basal 30 mm of the two innermost leaves of tillers were split longitudinally along the midvein and surface sterilized for 2 min in a 2.62% NaOCl solution containing 0.1% w/v Triton-X. The leaf halves were then washed in three changes of sterile distilled water. Each leaf half was cut transversely into six equal segments. The segments from one half of each leaf were serially explanted onto Petri plates containing Schenk and Hildebrandt [10] medium without growth regulators (SH0). Corresponding segments from the remaining half were plated onto the same medium but amended with 30 mM of the synthetic auxin 3,6-dichloro-2methoxybenzoic acid, also known as dicamba (SH30). The pH of SH0 and SH30 media was adjusted to 5.8 with 1 M NaOH prior to autoclaving. The leaf segments were then incubated in the dark at 22 °C for 4, 11, 17 or 28 days. The basal three segments of each treatment were collected separately from the distal three segments and then stored at −80 °C until total RNA isolation.
2.2. RNA isolation and mRNA differential display Total RNA was isolated using an RNeasy Kit (Qiagen, Valencia, CA), according to the manufacturer’s instructions. The RNAimage Kit (GenHunter, Nashville, TN) was used for RT-PCR differential display [11]. Three one-base-anchored oligo-dT primers, H-T11G, H-T11A, and H-T11C in combination with eight arbitrary primers, H-AP49 through H-AP56 (Table 1) were used for PCR amplification according to the manufacturer’s protocol. This method was first devised for human cells with the assumption that every cell expresses 15 000 genes [12]. To display all genes expressed, the method requires at least 25 arbitrary primers to achieve a level of confidence of about 0.95. Assuming that orchardgrass cells express the same number of genes, we can speculate that our study covered one third of them. The amplified cDNA frag-
ments labeled with 33P-dCTP were separated in a denaturing 6% polyacrylamide gel. The fragments were then visualized on a Kodak Biomax MS film by autoradiography. RT-PCR differential display was repeated three times for the reactions that produced differentially expressed cDNAs. Only bands that appeared repeatedly in the three independent reactions were selected, extracted from the gel and reamplified in a PCR reaction, as described by the kit manufacturer. The reamplified cDNA from each band was cloned in a pCR2.1 vector. Plasmids containing a DNA fragment were isolated from randomly selected bacterial colonies and then sequenced.
2.3. cDNA library construction and screening Basal leaf segments cultured on SH30 for 4 and 11 days, and basal and distal leaf segments cultured on SH30 for 25 days were used for mRNA isolation. A unidirectional cDNA library was constructed with the pooled mRNA in the Uni-ZAP XR vector (Stratagene, La Jolla, CA). The cDNAs were size fractionated prior to vector insertion. Fraction no. 1 contained the largest cDNAs and was used for screening. A minimum of twenty 150-mm agar plates with 50 000 pfu/plate was used for each screening. Plaque lifts were done with 132 mm discs of Hybond-N (Amersham, Piscataway, NJ). Probes were prepared by PCR amplification of a cDNA fragment under the same conditions as described for differential display, only with different nucleotide concentrations. Twenty micromolar each of dATP, dGTP, and dTTP, 10 mM of dCTP, and 5 ml of 10 mCi/ml [a-32P]dCTP were used in a 50 ml reaction. Five microliter of 1:100 dilution of the first-round PCR sample was used as a template. Membranes were hybridized in HYB-9 hybridization solution (Gentra, Minneapolis, MN) at 65 °C and then washed with a medium stringency wash solution (0.2× HYB-9) for approximately 2 h at 65 °C. Plaques that produced a hybridization
Table 1 Three anchored and eight arbitrary primers used for differential display of orchardgrass cultures Primers
Primer sequence (5%–3%)
H-T11G H-T11A H-T11C H-AP49 H-AP50 H-AP51 H-AP52 H-AP53 H-AP54 H-AP55 H-AP56
AAGCTTTTTTTTTTTG AAGCTTTTTTTTTTTA AAGCTTTTTTTTTTTC AAGCTTTAGTCCA AAGCTTTGAGACT AAGCTTCGAAATG AAGCTTGACCTTT AAGCTTCCTCTAT AAGCTTTTGAGGT AAGCTTACGTTAG AAGCTTATGAAGG
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signal were selected, and purified. Phagemid excision from the Uni-ZAP XR vector was done in vivo with ExAssist helper phage (Stratagene, La Jolla, CA) according to the manufacturer’s protocol.
2.4. RNA blot analysis Ten mg of total RNA from each RNA preparation were loaded onto a 1% agarose gel with formaldehyde and then blotted onto a Hybond-N membrane (Amersham, Piscataway, NJ). The membranes were pre-hybridized at 65 °C for 2 h in a solution containing 5% SDS, 0.33 M of sodium phosphate, 0.1 M of EDTA and 100 mg/ml salmon sperm DNA. Probes were synthesized with the RadPrime DNA labeling system (Gibco BRL, Grand Island, NY). An actin probe was made from a 500 bp actin fragment after EcoRI digestion of an Act5 clone. This clone contained the first actin exon from Lotus japonicus L., inserted in the pCR2.1-TOPO vector (Invitrogen, Carlsbad, CA). The membranes were washed at 65 °C with 2× SSC + 1% SDS for 1 h and with 0.1 × SSC + 0.5% SDS for an additional 1 h. They were then exposed to Kodak Biomax MS films for 10 – 14 days.
2.5. DNA blot analysis Genomic DNA was isolated from leaves using the Puregene DNA isolation kit (Gentra, Minneapolis, MN). Ten microgram of DNA was digested with HindIII, XhoI, BglII or BamHI, separated on a 0.8% agarose gel and blotted onto Hybond-N membranes (Amersham, Piscataway, NJ). The blots were pre-hybridized at 65 °C in HYB-9 hybridization solution (Gentra, Minneapolis, MN). Hybridization was performed with random primed 32P-labeled probes at 65 °C overnight. The membranes were then washed with a medium stringency 0.2× HYB-9 solution at 65 °C.
2.6. Sequencing and analysis DNA sequencing was performed with the ABI Prism Dye Terminator Cycle Sequencing reaction kit on an ABI 373 DNA sequencer (Perkin– Elmer, Foster City, CA). The initial sequence data text files were edited following comparison with the same data displayed in four-color electrophoregrams before they were analyzed further. Sequence data were analyzed using the Baylor College of Medicine (BCM) Search Launcher and the Expert Protein Analysis System (ExPASy) proteomics server of the Swiss Institute of Bioinformatics. Database searches were conducted with the basic local alignment search tool (BLAST) of the National Center for Biotechnology Information (NCBI).
Fig. 1. RT-PCR differential display of distal leaf segments after 28 days of culture. RT was performed with primer H-T11A (A), and H-T11C (B). PCR was carried out with the same anchored primer in combination with H-AP52 (A) and H-AP55 (B). Lane 1-Nonembryogen on SH0, lane 2-Nonembryogen on SH30, lane 3-Embryogen-P on SH0 and lane 4-Embryogen-P on SH30. Isolated bands marked with an arrow.
3. Results Because of the much lower response of distal leaf segments (a high proportion of the cells have already undergone differentiation), total RNA was isolated from these segments of Nonembryogen and Embryogen-P after 28 days culture on SH0 or SH30 medium. This RNA was used for RT-PCR differential display and their cDNA patterns were compared. Bands that appeared only in Embryogen-P cultured on SH30 medium were selected in order to avoid the cDNAs whose expression was induced by dicamba, but that was not related to somatic embryogenesis. Thus, eight cDNA bands were isolated from 24 reactions, but only two were used for further analyses (Fig. 1). Fragments D28.A52 and D28.C55b were 332 and 252 nucleotides in length, respectively, and represented the 3% end of two distinct mRNAs (data not shown). They showed
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no significant similarity with sequences in the NCBI GeneBank database. To determine the complete sequences of the differentially expressed cDNAs, fragments D28.A52 and D28.C55b were used as probes to screen a cDNA library of basal and distal embryogenic leaf cultures. Three independent clones were isolated from the cDNA library with the cDNA fragment D28.A52. The largest clone, DGE1 [accession no. AY011121] was 1733 nucleotides. Another clone, was only 5 bp shorter than DGE1 at the 5% end and was therefore named DGE1a. The third clone isolated with the same fragment was 1001 bp and was identical to the 3% end of DGE1. It was designated as DGE1b. The DGE1 ORF translated into a 386 aa polypeptide (Fig. 2). A BLAST homology search showed that a 90 aa region of DGE1 had 81% identity with the WRKY DNA-binding protein 21 from Arabidopsis thaliana (Fig. 3). The polypeptide sequence in the region of similarity contained an entire WRKY domain that is characteristic of the WRKY superfamily of plant transcription factors [13]. DGE1 contained glutamine-rich (180– 197) and serine-rich (222– 265) regions (Fig. 2), which are also characteristic of the WRKY proteins. Additionally, there was 87.9% identity between 1000 nucleotides at the 3% end of DGE1, part of which encoded the WRKY domain, and a clone
Fig. 2. DGE1 cDNA sequence and translation of the longest ORF. A stop codon is marked with a ‘*’. Glutamine-rich (180 –197) and serine-rich (222 – 265) regions are highlighted. The nuclear localization signal is underlined.
Fig. 3. Comparison of a segment from DGE1 containing the WRKY domain with other WRKY proteins. The alignment includes the amino acid sequence of WRKY protein 21 from Arabidopsis thaliana [accession no. AF272747], WRKY3 from A6ena sati6a L., oat [accession no. AF140553], WRKY transcription factor Nt-SubD48 from Nicotiana tabacum, tobacco [accession no. AB041520], and WRKY3 from Petroselinum crispum L, parsley [accession no. U56834]. Location of identical amino acids in at least four organisms is highlighted.
from a 4–45 days after pollination spike EST library of barley (Hordeum 6ulgare L.) [accession no. BE194160]. Furthermore, a 78.7% identity was found over 361 nucleotides starting at position 140 with a cDNA from rice (Oryza sati6a L.) etiolated leaf sheath [accession no. AI978441]. Analyses of the DGE1 polypeptide suggested that it was most likely a soluble protein with a theoretical pI of 9.97 and a relative molecular mass of 41 700. A nuclear targeting sequence was predicted by the PSORT/ExPaSy at position 297–300 (Fig. 2). DGE2 [accession no. AY011122] was isolated from the cDNA library with fragment D28.C55b. It had 1378 nucleotides with an ORF that encoded a 354 aa polypeptide (Fig. 4). Searches of the GenBank/NCBI databases showed that a 423 nucleotide segment of the clone, starting at residue 182, was 47.8% identical to a sorghum (Sorghum bicolor L.) pathogen induced PI1 clone [accession no. BE600715]. Furthermore, a 79.5% identity with a clone from a wheat (Triticum aesti6um L.) pre-anthesis spike cDNA library [accession no. BE500740] was exhibited over 410 nucleotides starting at position 624. DGE2 showed no significant similarity with DGE1. The primary sequence analyses of DGE2 showed that it was likely a soluble protein with a pI of 10.1 and a molecular mass of 41 500. The polypeptide had a high, approximately 20%, arginine content. Three nuclear targeting sequences were predicted by the PSORT/ExPaSy Molecular Biology Server. Two are K and R rich regions and the third is known as the Robbins and Dingwall consensus [14], which is defined by two basic residues, a ten residue spacer, and another basic region consisting of at least three basic residues out of five. The differential expression of the isolated clones was confirmed by RNA blot analysis (Fig. 5). Low expression levels of DGE1 were observed in basal leaf segments at 4, 11, 17 and 28 days, and also in distal leaf segments at 17 and 28 days of culture on SH30 medium. DGE1 produced no hybridization with DNA
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from leaf segments cultured on SH0 medium. The hybridization signal produced by DGE2 was limited to distal leaf segments cultured on SH30 for 28 days. Genomic DNA separately digested with HindIII, XhoI, BglII or BamHI was subjected to DNA hybridization with the cDNAs for DGE1 or DGE2 as a probe. Two or three hybridizing bands were found in each lane under a medium-stringency condition (Fig. 6). This is an indication that there are probably two DGE1-related and two DGE2-related genes in Embryogen-P.
4. Discussion Embryogenic orchardgrass leaf cells are located primarily in the leaf mesophyll subjacent to both the adaxial and abaxial leaf surfaces [15]. The initial divisions are observed as early as 4 days after culture initiation. Approximately 70– 80% of the initial divisions are periclinal to the leaf surface [16]. Divisions of the daughter cells occur 6– 8 days after initiation of cultures. The culture time necessary for initiation of the first divisions corresponds to the earliest time of detection of somatic embryogenesis receptor-like kinase (SERK) transcripts [17]. SERK was proposed to be a marker for cells competent to form embryos [8]. In the present study, a RT-PCR differential display technique [11] was used to identify genes that were
Fig. 4. DGE2 cDNA sequence and translation of the longest ORF. A stop codon is marked with a ‘*’. Predicted nuclear targeting K and R rich sequences are underlined and a sequence matching the Robbins and Dingwall consensus for a bipartite nuclear localization signal is marked with a double line.
Fig. 5. RNA blot analyses of DGE1 and DGE2. An actin clone Act5 from Lotus japonicus was used as a control. Ten milligram total RNA from Embryogen-P was loaded in each lane. RNA was isolated from basal and distal leaf segments cultured for 4, 11, 17, or 28 days on SH0 or SH30 medium. Embryogenic capacity was measured as a number of embryos developed into plantlets after induction of leaf segments on SH30 medium for 4, 11, 17 or 28 days, followed by culture on SH0 for 30 days.
expressed during direct or indirect embryogenesis. This method is advantageous to the numerous variations of subtractive hybridization procedures because the expression of more than two sets of samples can be monitored simultaneously. Four sets of samples were compared that represented three different controls and one sample in which somatic embryogenesis occurred. The expression patterns were compared for leaf segments from Embryogen-P and Nonembryogen cultured on SH0 or SH30. Presumably, Nonembryogen leaf segments responded to dicamba in the same way as Embryogen-P except that embryogenesis was not induced. Therefore, Nonembryogen was used to eliminate the genes induced by dicamba, but not involved in the process of somatic embryogenesis. In total, 24 pairs of primer combinations were used and eight bands were identified which were induced by dicamba in embryogenic leaf cultures. The cDNA from two of the bands was used to prepare PCR probes for library screening. Thus, two unique clones, DGE1 and DGE2, were isolated from the cDNA library, that corresponded to mRNAs induced during somatic embryogenesis. The DGEs were differentially expressed in embryogenic tissues. Low levels of DGE1 transcripts were detected in embryogenic basal segments over the entire course of study and distal leaf segments starting at 17 days. This suggested that DGE1 might play a role in
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indirect and direct embryogenesis, or alternatively is a reflection of the basal leaf cell forming direct embryos. Distal leaf segments after 17 days culture on SH30 medium, produced only a few embryos, but more leaf cells might have been competent for embryogenesis at that time. The expression of DGE1 in these segments implied that it might have a role in the induction of embryogenesis. The DGE2 probe only hybridized to RNA from embryogenic distal leaf segments cultured for 28 days on SH30. This suggested that DGE2 may not be expressed in embryogenic basal leaf segments and therefore, indirect embryogenesis in basal leaf and direct embryogenesis in the more distal leaf cells might follow different pathways. Neither DGE1 nor DGE2 transcripts were detected in RNA from nonembryogenic leaf segments cultured on SH0 medium. DGE1 encodes a 386 aa polypeptide predicted to be a soluble protein with a pI of 9.97 and to have a nuclear targeting sequence. A high similarity was found between the C-termini of DGE1 and WRKY21 from Arabidopsis that contained a WRKY domain and a nuclear targeting sequence. WRKY21 is a member of the WRKY superfamily of sequence-specific DNAbinding proteins that have been found only in plants. This is a family of transcriptional regulators involved in senescence and trichome development [13]. Furthermore, they play a key role in a signal transduction pathway that leads from elicitor perception to the activation of pathogenesis-related genes from class 1 [18].
These facts suggest that DGE1 resides primarily in the nucleus and might have a transcriptional regulatory function. In addition, the accumulation of three PR proteins was correlated with somatic embryogenesis [19]. Therefore, the partial similarity of DGE1 to a gene involved in response to pathogens suggested that similar proteins are involved in the induction of somatic embryogenesis and PR proteins. The putative DGE2 protein shared certain features with that of DGE1. It was a hydrophilic protein with a pI of 10.1 that contained putative nuclear targeting sequences. However, no sequence homology was found between DGE1 and DGE2. The sequence similarity of one part of DGE2 with a pathogen induced clone PI1 from Sorghum bicolor and another part with a clone from a wheat pre-anthesis spike cDNA library suggested that DGE2 might share domains with other proteins, but their role remains unknown. In conclusion, two genes, DGE1 and DGE2 were identified to be associated with somatic embryo formation in orchardgrass. These two genes are likely to encode nuclear proteins with possible regulatory functions and may be responsible for some of the mechanisms essential for the formation of embryogenic plant cells, especially in Poaceae. Acknowledgements The authors thank Dr Neil Quigley from the Molecular Biology Resource Facility at UTK for the DNA sequencing, and Drs Albrecht von Arnim and Beth Mullin from the Department of Botany at UTK for their support and assistance in the preparation of the manuscript. We also thank Ledare Habera for professional guidance throughout the project. References
Fig. 6. Genomic DNA blot analysis of DGE1 and DGE2. Ten microgram DNA was digested with HindIII (H), XhoI (X), BglII (Bg) or BamHI (Ba). After blotting the DNA was probed with cDNAs from DGE1 or DGE2. Both DGE1 and DGE2 clones lack restriction sites for the enzymes used. Sizes of DNA markers are indicated on the left.
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