The scutellar vascular bundle–specific promoter of the wheat HD-Zip IV transcription factor shows similar spatial and temporal activity in transgenic wheat, barley and rice Nataliya Kovalchuk, Wei Wu † , Omid Eini, Natalia Bazanova, Margaret Pallotta, Neil Shirley, Rohan Singh, Ainur Ismagul, Serik Eliby, Alexander Johnson ‡ , Peter Langridge and Sergiy Lopato* Australian Centre for Plant Functional Genomics, University of Adelaide, Hartley Grove, Urrbrae, South Australia, Australia Received 1 March 2011; revised 22 April 2011; accepted 3 May 2011. *Correspondence (Tel +61 8 830 37 499; fax +61 8 830 37 102; email sergiy.lopato@acpfg.com.au) † Present address: Agronomy College, Sichuan Agricultural University, Yaan 625014, China. ‡ Present address: School of Botany, The University of Melbourne, VIC 3010, Australia. Accession numbers: TaGL9—JF332037; TdGL9H1—JF332038; TdGL9H2—JF332036. Keywords: HD-Zip IV transcription factor, promoter, embryo, scutellar vascular bundle, wheat, barley, rice. Summary An HD-Zip IV gene from wheat, TaGL9, was isolated using a Y1H screen of a cDNA library prepared from developing wheat grain. TaGL9 has an amino acid sequence distinct from other reported members of the HD-Zip IV family. The 3¢ untranslated region of TaGL9 was used as a probe to isolate a genomic clone of the TaGL9 homologue from a BAC library prepared from Triticum durum L. cv. Langdon. The full-length gene containing a 3-kb-long promoter region was designated TdGL9H1. Spatial and temporal activity of TdGL9H1 was examined using promoter-GUS fusion constructs in transgenic wheat, barley and rice plants. Whole-mount and histochemical GUS staining patterns revealed grain-specific expression of TdGL9H1. GUS expression was initially observed between 3 and 8 days after pollination (DAP) in embryos at the globular stage and adjacent to the embryo fraction of the endosperm. Expression was strongest in the outer cell layer of the embryo. In developed wheat and barley embryos, strong activity of the promoter was only detected in the main vascular bundle of the scutellum, which is known to be responsible for the uptake of nutrients from the endo- sperm during germination and the endosperm-dependent phase of seedling development. Furthermore, this pattern of GUS staining was observed in dry seeds several weeks after harvesting but quickly disappeared during imbibition. The promoter of this gene could be a useful tool for engineering of early seedling vigour and protecting the endosperm to embryo axis pathway from pathogens during grain desiccation and storage. Introduction In many eukaryotic organisms, including higher plants, homeod- omain (HD, known also as homeobox) transcription factors are important regulators of embryo development (Lawrence and Morata, 1994; Mukherjee et al., 2009). Plant HD transcription factors contain a 60-amino acid-long conserved sequence and were originally divided into five families: HD-ZIP, GLABRA, KNOTTED, PHD and BEL (Chan et al., 1998). One of the largest families, homeodomain-leucine zipper (HD-Zip) transcription factors (TFs), comprises four classes of proteins (class I–IV). This family contains 41 genes in rice and 70 genes in maize (Mukherjee et al., 2009). Class IV HD-Zip (HD-Zip IV, also called in literature as HD-GL2 family) TFs have four well-defined domains: the DNA-binding HD, leucine zipper domain (for dimerization), a steroidogenic acute regulatory protein-related lipid transfer (START) domain (Ponting and Aravind, 1999; Tsujishita and Hurley, 2000) and a START-associated conserved domain (HD-SAD) (Schrick et al., 2004; Mukherjee and Burglin, 2006). The role of the START and SAD domains is still unclear. However, it was demonstrated that they are involved in interactions with the transcription initi- ation complex and other proteins. The transcriptional activation domain of ZmOCL1 was localized to 85 amino acids in the N-terminal part of the START domain, and interaction between OCL1 and SWI3C1 (a subunit of the SWI ⁄ SNF chromatin remodelling complex) was demonstrated (Depege-Fargeix et al., 2011). It was recently reported that the HD-SAD domain of GbML1 is required for the interaction with an R2R3-type MYB factor, GbMYB25 (Zhang et al., 2010). It was shown in vitro that the HD-Zip proteins bind to 9-bp- long palindromic sequences (Sessa et al., 1993, 1998; Tron et al., 2001). Later, it was demonstrated that Arabidopsis ATML1 and PDF2 proteins bind to repeats of the L1 box TAAATG(C ⁄ T)A (Abe et al., 2001, 2003). Another class IV protein from Arabidopsis, HDG7, showed a binding preference for the palindromic sequence GCATTAAATGC that partially overlaps with the L1 box (Nakamura et al., 2006). The HDG9 binding sequence GCATTAAATGCGCA contains the HDG7 binding sequence and also an L1 box-like motif (Nakamura et al., 2006). Sixteen genes encoding HD-Zip IV proteins were identified in Arabidopsis (Nakamura et al., 2006). Many of them were shown to specify cell fate in outer cell layers during early embryogenesis and at early developmental stages of plant meristem (Nakamura et al., 2006). Members of HD-Zip IV demonstrate the specific expression patterns that are different from other HD proteins. In Arabidopsis, the GL2 gene is expressed in the outer cell layers of shoots and roots (Rerie et al., 1994; DiCristina et al., 1996), and ATML1 is expressed in meristems throughout the diploid life cycle (Lu et al., 1996; Sessions et al., 1999). ª 2011 Australian Centre for Plant Functional Genomics, University of Adelaide Plant Biotechnology Journal ª 2011 Society for Experimental Biology, Association of Applied Biologists and Blackwell Publishing Ltd 43 Plant Biotechnology Journal (2012) 10, pp. 43–53 doi: 10.1111/j.1467-7652.2011.00633.x