Cloning of a cDNA encoding EIN3‐like protein (DC‐EIL1) and decrease in its mRNA level during senescence in carnation flower tissues

Abstract

A cDNA clone encoding a putative EIN3-like protein (DC-EIL1) was obtained from total RNA isolated from senescing carnation (Dianthus caryophyllus L.) petals using RT-PCR and RACE techniques. The cDNA (2382 bp) contained an open reading frame of 1986 bp corresponding to 662 amino acids. The amino acid sequence of the N-terminal half of the protein, ranging from 80-300 amino acid residues, had 84% identity with that of the corresponding regions of Arabidopsis EIN3 and tobacco TEIL, although the overall identity was 49% and 52%, respectively. Northern blot analysis revealed that the amount of mRNA corresponding to DC-EIL1 decreased in flower tissues, especially in petals, during natural senescence and senescence induced by exogenously applied ethylene or ABA.

Flower senescence: some molecular aspects

“…The transcripts encoding a range of transcription factors have been isolated and found to be differentially regulated during development and senescence in various flower systems (Table 5), e.g., a homeodomain protein (a class of proteins generally representing transcription factors), MYBlike DNA-binding protein, MYC protein and Zinc-finger protein from Dianthus and Mirabilis, MADS Box genes from carnation, Iris and Arabidopsis, NAC domain transcription factors and CeBP "Carnation ethylene-responsive element binding protein" (waki et al 2001;Fang and Fernandez 2002;van Doorn et al 2003;Hoeberichts et al 2007;Iordachescu et al 2009;Balazadeh et al 2010). The Iris MADS box gene has been found to share 51 % identity with RIN (a MADS-box factor involved in developmental control of fruit ripening in tomato; vrebalov et al 2002) and that the corresponding translated fragment from carnation shares 34 % identity with tomato RIN and 55 % identity with the Arabidopsis pistillate protein (Hoeberichts et al 2007).…”

“…Perception of ethylene is brought about by a family of eTRs that in turn regulates the activity of CTR1 (a negative regulator of the ethylene response pathway) whose protein sequence have been reported to share similarity to the Raf family of serine/threonine protein kinase thereby suggesting that it (CTR1) might act via Mitogen-activated protein (MAP) kinase cascade, since MAPKs have been have been implicated in coordinating stress responses, probably as the key factors in the PCD signal transduction pathway (Kieber et al 1993;Mizoguchi et al 1996;waki et al 2001). In Delphinium, the DlCTR1, encoding a polypeptide of 800 amino acids containing the expected serine/threonine kinase domain, the consensus ATP-binding site, and the serine/threonine kinase catalytic site has also been characterized (Kuroda et al 2004).…”

“…Recently, a carnation cDNA (DCEBF1; 1,878 bp) encoding eBF-like protein has been isolated whose expression has been reported to be enhanced by endogenous/exogenous ethylene, and inhibited by STS in petals and ovaries (Fu et al 2011b). EIN3 or EIN3-like proteins (EIL1, EIL2, EIL3, EIL4 and EIL5; nuclear-localized transcription factors) have been found to be upregulated during senescence (waki et al 2001;Alonso et al 2003;Hoeberichts et al 2003;Yanagisawa et al 2003;Shibuya et al 2004;Iordachescu and verlinden 2005;Zhou et al 2010). As far as structural aspect of Arabidopsis EIN3 protein is concerned, it has been found to harbor a highly Kuang et al (2012) acidic domain at N-terminus, five small clusters of basic amino acids throughout the EIN3 polypeptide, a proline rich domain, and an asparagine-rich domain at the C-terminus (Chao et al 1997).…”

Cloning, Characterization and Expression of Carnation (Dianthus caryophyllus L.) Ubiquitin Genes and Their Use as a Normalization Standard for Gene Expression Analysis in Senescing Petals

“…no. Y0073415, Waki et al, 2001), and DcUbqs was conducted using the LightCycler FastStart DNA Master SYBR Green I Kit and the LightCycler Instrument (Roche Diagnostics, Tokyo, Japan). cDNA was synthesized using ReverTra Ace (TOYOBO, Osaka, Japan) according to the manufacturer's instructions.…”

“…So far, rRNA has been frequently used as a standard gene for studying the expression of genes relevant to senescence in carnation flowers (Hoeberichts et al, 2007;Iordachescu and Verlinden, 2005;Jones, 2002;Van der Kop et al, 2003;Verlinden and Garcia, 2004;Verlinden et al, 2002). In addition, actin (DcACT1) transcript has been used as the standard gene for normalizing sample RNAs instead of rRNA Nukui et al, 2004;Shibuya et al, 2002;Sugawara et al, 2002;Waki et al, 2001).…”