Characterization of CsSEF1 gene encoding putative CCCH-type zinc finger protein expressed during cucumber somatic embryogenesis
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) (Wróblewski 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 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.
Section snippets
Plant materials
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 Wróblewski 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
Characterization of CsSEF1 sequence
Differential screening of the SSH library involved hybridization of arrayed bacterial colonies with 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
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 proteins containing the classical CCHH zinc
Acknowledgments
This work was supported by Polish Ministry of Science and Higher Education (grant no. PBZ/KBN/029/P06/2000).
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These authors shared equal participation in this work.