The conserved ELK-homeodomain of KNOTTED-1 contains two regions that signal nuclear localization

Abstract

Nuclear localization serves as a regulatory mechanism in the activity of several transcription factors. KNOTTED-1 (Kn1) is a homeodomain protein likely to regulate vegetative development in maize. At least twelve genes related to Kn1 are known in maize and six in Arabidopsis. Ectopic expression of the maize, rice and Arabidopsis Kn1-related genes have been shown to alter cell fate determination. In this paper, we study the nuclear localization capabilities of the Kn1 homeodomain and the proximal amino acid residues (the ELK region) which is highly conserved among Kn1-related homeodomain proteins. The ELK homeodomain (ELK-HD) of Kn1 was fused to the reporter gene uidA encoding the bacterial enzyme beta-glucuronidase (GUS) and transformed into tobacco and onion cells. Quantitation of GUS activity in nuclear and total protein extracts from transgenic tobacco revealed a highly localized GUS activity in the nucleus for the ELK-HD/GUS fusion protein, as compared to the basal level of GUS activity in the nucleus for the GUS only protein. The ELK-HD/GUS transformants showed no unusual characteristics, thus indicating that expression of the putative Kn1 DNA-binding domain fused to GUS may be insufficient to create a dominant negative phenotype. Histochemical analysis of the onion epidermal cells transfected by particle bombardment demonstrated that greater than 50 % of the transformed onion epidermal cells showed higher levels of GUS staining in the nucleus relative to the cytoplasm. Deletion analysis of the ELK-HD revealed that the Kn1 homeodomain comprising the three predicted alpha-helices and the conserved ELK domain can each function independently as nuclear localization signals.

A Rapid and Efficient Method for Purifying High Quality Total RNA from Peaches (Prunus persica) for Functional Genomics Analyses

“…The second protocol is based upon a phenol:chloroform extraction and LiCl precipitation protocol as describe by Das et al (1990). These protocols have been used to isolate RNA from leaf tissue in model plants such as arabidopsis, tobacco and maize (Das et al, 1990;Meisel and Lam, 1996;Silva et al, 1999). However, use of such protocols on peach fruits yielded poor quality RNA with high levels of polysaccharides, polyphenolic compounds, and protein contaminants as determined by A240/260 and A260/280, respectively (Logemann et al, 1987;Manning 1990) (Table I).…”

Nuclear Localization of NPR1 Is Required for Activation of PR Gene Expression

“…To facilitate identification of the functional NLS or NLSs, we used a transient assay involving the biolistic bombardment of various NPR1-GFP fusion constructs into onion epidermal cells. This assay is a quick and effective means of identifying the NLSs in a variety of plant proteins (Varagona et al, 1992;Meisel and Lam, 1996; van den Ackerveken et al, 1996). On the basis of the constitutive expression of the BGL2::GUS reporter gene detected in this assay, the onion cells appear to represent the SARinduced state (data not shown).…”

Transcriptional, Posttranscriptional, and Posttranslational Regulation of SHOOT MERISTEMLESS Gene Expression in Arabidopsis Determines Gene Function in the Shoot Apex

“…The HD is required for DNA binding (Smith et al, 2002;Viola and Gonzalez, 2009) and intercellular trafficking (Lucas et al, 1995;Kim et al, 2005;Winter et al, 2007b;Bolduc and Hake, 2009). However, the role of the ELK domain is less understood, although it is considered to be required for protein-protein interaction (Vollbrecht et al, 1991;Kerstetter et al, 1994) and for nuclear localization signal (NLS) sequences (Meisel and Lam, 1996). Cole et al (2006) indicated that STM does not contain an NLS, and in the rice KNOX1 gene OSH15, the ELK domain is not required for nuclear localization, DNA binding, or homodimer formation, although it was shown to have a role in suppressing transactivation activity (Nagasaki et al, 2001).…”