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 endosperm 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.
In many eukaryotic organisms, including higher plants, homeodomain (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 initiation 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).
It has been shown that the ZmOCL1, ZmOCL3, ZmOCL4 and ZmOCL5 genes of maize have similar expression patterns. They were detected in the upper cell layers of embryos at the 16-cell stage, the apical shoot meristem, inflorescence meristem, and floral or young organ primordia at later stages (Ingram et al., 1999, 2000). One exception was the expression of ZmOCL2, which was restricted to the subepidermal cell layers of meristems (Ingram et al., 2000). ZmOCL1 was overexpressed in transgenic maize and 14 genes were up- or down-regulated (Javelle et al., 2010). The downstream genes encoded proteins involved in lipid metabolism, defence and cuticle biosynthesis (Javelle et al., 2010).
The Norway spruce homeobox gene PaHB1 (Picea abies Homeobox1) demonstrated an expression pattern similar to some other reported members of the class IV HD-Zip TFs, a switch from ubiquitous to protoderm-specific localization during somatic embryo development (Ingouff et al., 2001). However, PaHB2, another HD-GL2 family member from P. abies, is evenly expressed in early somatic embryos but becomes preferentially localized in the outer cortical layer and root cap of mature embryos (Ingouff et al., 2003). PaHB2 was also detected in the same root tissues at post-embryonic stages of root development. The amino acid sequence of PaHB2 is very similar to the protein sequence of ANTHOCYANINLESS2 (ANL2) from Arabidopsis (Kubo et al., 1999). The anl2 mutant has aberrant radial root patterning: roots produce extra cells called intervening cells, located between the cortical and epidermal layers (Kubo et al., 1999). This suggests that the main role of ANL2 and PaHB2 may be maintenance of subepidermal cell layer identity.
The GL2-type homeobox gene from rice, Rice outermost cell-specific gene 1 (Roc1), is expressed mainly in the protoderm of the developing embryos, but is also localized to the L1-layer or protoderm of the SAM during shoot and flower development, in the outermost cells of the ligule and partially regenerated callus and in existing and newly produced cells at the cut ends of the callus (Ito et al., 2002). In contrast, another rice homeobox gene, OsTF1, encodes a very distinct member of the family that is expressed only in grain and young roots (Yang et al., 2002). In grain, it was detected everywhere in the developing embryo between 3 and 7 days after pollination (DAP) with the strongest expression in the upper cell layer; weak expression was also observed in the endosperm (Yang et al., 2002).
Most of the data about spatial expression patterns of HD-Zip IV transcription factors from grasses were obtained using in situ hybridization. Promoter analysis using fusions to the uidA reporter gene was used only for the members of Arabidopsis HD-Zip IV genes (Nakamura et al., 2006). Five of the 16 Arabidopsis genes demonstrated either specific or predominant expression in flowers and/or siliques (Nakamura et al., 2006).
In this study, a new HD-Zip IV gene from wheat, designated Triticum aestivum GLABRA2-like clone 9 (TaGL9), was isolated in a Y1H screen of a cDNA library prepared from wheat grain at 0–6 DAP. A close homologue of TaGL9 was isolated from a BAC library prepared from T. durum and designated as Triticum durum GL9 homologue 1 (TdGL9H1). Spatial and temporal expression patterns of TaGL9 and TdGL9H1 were examined by quantitative real-time PCR (Q-PCR) and revealed grain-specific expression of these genes. A promoter-GUS fusion construct was generated for TdGL9H1 and was used for stable transformation of wheat, barley and rice plants. Whole-mount and histochemical GUS staining patterns revealed grain-specific activity of TdGL9H1 promoter in transgenic plants. Expression was detected as early as 5 DAP in the outer layers of the embryo and adjacent to embryo part of the endosperm. Beyond 5 DAP, promoter activity was only observed in the main vascular bundle of the scutellum. No differences in TdGL9H1 promoter activity were observed between wheat and barley. However, TdGL9H1 promoter activity in rice was also detected in vascular bundles of embryonic coleoptiles and leaves.
Cloning and sequence analysis of GL9 genes
The full-length cDNA of TaGL9 was isolated from a Y2H cDNA library prepared from the whole grain of T. aestivum, cultivar Chinese Spring at 0–6 DAP. The bait DNA sequence used to screen the cDNA library consisted of four tandem repeats of the cis-element, CATTAAATG, which is specific for HD-Zip IV TFs (Abe et al., 2001). Of 48 positive clones, seven contained inserts longer than 2 kb. Two positive clones contained a 2.6-kb insert, while five clones contained a 3.3-kb insert. Sequencing revealed that the differently sized inserts encoded two different cDNAs containing full-length coding regions of HD-Zip IV TFs. One of them, designated TaGL9, was 2.6 kb long. The coding region of GL9 was recloned in frame with the yeast activation domain (AD), and specific interaction with a bait sequence was confirmed in the Y1H assay (Figure 1). A database search using the TaGL9 protein sequence revealed the closest annotated homologue to be OsTF1 (Acc. AF317882) that shared 46.2% identity (Yang et al., 2002). Southern blot hybridization of nullisomic-tetrasomic lines of hexaploid wheat (Sears, 1954) with 3′ UTR of TaGL9 as probe revealed that TaGL9 is located on group 3 chromosomes of hexaploid wheat. A 172-bp-long fragment of 3′ untranslated region (3′ UTR) of TaGL9 was used as a probe to screen a bacterial artificial chromosome (BAC) library prepared from genomic DNA of T. durum cv. Langdon (Cenci et al., 2003), using Southern blot hybridization. Five BAC clones were identified; BAC DNA was isolated from the three that demonstrated the strongest hybridization with the probe and used as a template for PCR with primers derived from the coding region and 3′ UTR of TaGL9. Only one of the BAC clones gave a PCR product. The 454 sequencing of this clone revealed a full-length gene (9 kb, including promoter sequence) of a close homologue/homoeologue of TaGL9 from T. durum, which was designated TdGL9H1. The coding region of the cloned gene is interrupted with 10 introns. The coding region of the one more cDNA, designated TdGL9H2, was isolated from a cDNA pool prepared from developing grain using primers derived from the genomic sequence of TdGL9H1. The deduced protein sequences of TdGL9H1 and TdGL9H2 have 81.9% and 87.2% identity, respectively, with TaGL9 and 46.5% and 46.8% identity, respectively, with OsTF1. The interaction of TdGL9H2 with repeats of the CATTAAATG element was confirmed in a Y1H assay (Figure 1). A cis-element specific to HD-Zip class II transcription factors, CAATGATTG, was used as a negative control. In contrast to the AD of the yeast GAL4 transcription factor expressed from the empty pGADT7 vector (negative control), both TaGL9 and TdGL9H2 strongly interacted with the CATTAAATG sequence and did not interact with the negative control (Figure 1). Weak activation of the reporter genes was detected if the full-length TaGL9 was expressed in frame with the BD of the yeast GAL4 transcription factor, suggesting the possible presence of an activation domain in TaGL9 (data not shown).
The phylogenetic relationships based on amino acid sequences of TaGL9, TdGL9H1, TdGL9H2 and the sequences of the HD-Zip class IV proteins from other plant species are shown in Figure 2a. The closest homologue of GL9 proteins is OsTF1 from rice. An alignment of TaGL9 to protein sequences of TdGL9H1, TdGL9H2 and OsTF1 is shown in Figure 2b. The closest homologues of GL9 proteins from Arabidopsis, AtHDG8, AtHDG9, AtHDG10 and FWA/AtHDG6, share about 30% amino acid sequence identity with wheat GL9 proteins. Genes encoding these four Arabidopsis proteins were found to be expressed only in flowers and/or siliques (Nakamura et al., 2006). This suggests the possible presence of a larger number of grain-specific homologues of GL9 genes in wheat.
Computer analysis of the 3029-bp-long TdPR9H1 promoter fragment using the PLACE database (http://www.dna.affrc.go.jp/PLACE/signalscan.html) to identify potential plant cis-acting regulatory DNA elements (Higo et al., 1999) revealed a large number of cis-elements that are known to be responsible for the specific gene expression in endosperm, embryo and seeds. The promoter contains a putative prolamin box, TGCAAAG, which was earlier found in the rice GluB-1 promoter and is involved in quantitative regulation of endosperm-specific genes (Wu et al., 2000). Another quantitative element identified in the TdGL9H1 promoter is the RY repeat motif, CATGCATG, which is responsible for seed-specific expression of many genes in both dicotyledonous and monocotyledonous plants (Baumlein et al., 1992; Thomas, 1993; Curaba et al., 2004). The binding site of the embryo-specific bZIP transcription factors, ACACNNG (Kim et al., 1997), is repeated eight times in the TdGL9H1 promoter. The promoter also contains multiple E-boxes, CANNTG, which usually act together with ABRE elements and are responsible for seed-specific expression (Stalberg et al., 1996; Hartmann et al., 2005).
In addition to seed-specific cis-elements, the TdGL9H1 promoter is enriched for putative sugar responsive elements. One such element, the S-box, CACCTCCA, was found to be conserved in several rbcS promoters in Arabidopsis (Acevedo-Hernandez et al., 2005). Another cis-element, responsible for sugar-regulated repression, is an A-box or G motif, TACGTA, and is a binding site for bZIP factors (Toyofuku et al., 1998). A putative pyrimidine box (CCTTTT) that was also reported to be partially responsible for sugar-mediated repression (Morita et al., 1998) is repeated four times in the TdGL9H1 promoter.
Spatial and temporal patterns of TaGL9 and TdGL9H1 expression
Expression of TaGL9 and TdGL9H1 in different wheat (T. aestivum cv. Chinese Spring and T. durum cv. Langdon, respectively) tissues was analysed using Q-PCR. TaGL9 was found to be weakly expressed in anthers, mature embryos at 22 DAP and in the embryo and roots of germinating seedlings. Strong expression was detected in the caryopsis at 3–5 DAP and endosperm at 22 DAP (Figure 3a). Expression of TaGL9 in grain was detected at 4 DAP and was observed until 20 DAP. It decreased at 7 DAP (coinciding with filling of endosperm with starch), but later increased again to peak at 17–18 DAP (Figure 3b). No expression of TdGL9H1 was detected in all tested tissues except low level of expression in crown (Figure 3a). In grain, expression of TdGL9H1 has constantly increased, reached maximum at 11–15 DAP and then started to decrease (Figure 3b).
Activity of the TdGL9H1 promoter in the developing and mature grain of wheat, barley and rice
Spatial and temporal expression patterns of TdGL9H1 were examined using a promoter-GUS fusion construct and stable transformation of wheat, barley and rice plants. Whole-mount staining patterns and histochemical GUS staining patterns were analyzed in independent transgenic lines (Table 1). GUS staining was initially observed in wheat, barley and rice grains in areas surrounding embryos at 5 DAP. Embryos at this stage of development have a near-globular shape and initiation of the primordium of the seminal root, shoot apex and vascular bundle of the scutellum has just commenced (Figures 4a,j, 5a–d and 6a–d). Histochemical analysis of transgenic wheat and barley grains at 5–8 DAP revealed GUS expression in the embryo and endosperm; the strongest expression was observed in the epidermal cell layer of the embryo and the fraction of partially cellulorized endosperm adjacent to the embryo (Figures 5a–d, 6a–d and 7g). No expression was detected in the rest of the endosperm. In the developed embryo of wheat, barley and rice after 11–12 DAP, when multiplication of cells by cell division has almost ceased and development of the vascular bundle system is completed (Hoshikawa, 1989), strong activity of the promoter was detected only in the main vascular bundle of the scutellum (Figures 4b–i,m–s, 5e–i, 6e,f and 7b–f,h–l). Expression slowly increased in rice or remained the same in wheat and barley until grains had fully developed (Figures 4b–i,m–s and 7b–f). GUS activity was also detected in the main vascular bundle of the scutellum several weeks after the grain was harvested (Figure 4g–i). No GUS expression was detected in any other part of the embryo. Similarly to wheat, the activity of the promoter in transgenic rice plants was initially observed at 5 DAP in endosperm around the embryo (Figure 7a,g) and later, between 12 and 50 DAP, found only in the main vascular bundle of the scutellum (Figure 7b,d,g). Beyond 50 DAP, however, GUS staining also appeared in the shoots of embryos (Figure 7c, e, f, h,i–l). At 59 DAP, GUS expression was detected everywhere in the embryonic coleoptiles and leaves, with the strongest expression in vascular bundles and adaxial parts of coleoptiles (Figure 7f,k). This pattern of GUS staining in transgenic rice plants did not change until at least 69 DAP (Figure 7k) and remained in harvested grain. However, it quickly disappeared during imbibition and following germination (data not shown). No activity of the TdGL9H1 promoter has been detected in other tested tissues of wheat, barley and rice, including leaf, stem, root, meristems and different parts of flower (data not shown).
Table 1. Information about transgenic plants transformed with pTdGL9H1 construct
Number of transgenic lines
T0 lines selected on Hyg and confirmed by PCR
T0 lines tested for GUS activity
T0 lines with strong expression
T0 lines with weak expression
T0 lines with no expression
Sterile T0 lines
T0 lines with different pattern of expression
T1 plants tested for GUS activity
T1 plants with strong expression
T1 plants with weak expression
T1 plants with no expression
It was previously demonstrated that five of the 16 Arabidopsis HD-Zip IV genes are either specifically or predominantly expressed in flowers and/or siliques (Nakamura et al., 2006). Searching for early grain-specific promoters from wheat, we isolated cDNAs of two transcription factors using a Y1H screen of a grain-cDNA library with the palindromic sequence, CATTAAATG, that is specific for Arabidopsis HD-Zip IV TFs, repeated four times (Tron et al., 2001). This bait sequence also contained three repeats of the palindromic consensus GCATTAAATGC obtained in selection experiments with four recombinant Arabidopsis HD-Zip IV proteins (Nakamura et al., 2006). The L1-box sequence, TAAATGCA, an asymmetric cis-element previously identified in the promoters of several target genes from Arabidopsis was also repeated three times (Abe et al., 2001; Ohashi et al., 2003). One of the cloned transcription factors, TaGL9, contains all the domains characteristic of the HD-Zip IV TFs (Figure 2b); however, it shows low sequence similarity with most of the known members of this family (Figure 2a). A full-length gene and cDNA encoding a close homologues of TaGL9 were isolated from a durum wheat. TdGL9H1 and TdGL9H2 share 81.9% and 87.2% amino acid sequence identity with TaGL9. Specific interaction of both TaGL9 and TdGL9H2 with the bait DNA sequence used to isolate the clones from the Y1H assay was confirmed (Figure 1); the cis-element specific for the class II HD-Zip TFs was used as a negative control. Previous data (Tron et al., 2001; Nakamura et al., 2006) and our unpublished results suggest that there are few differences in the specificity of DNA binding between different members of the HD-Zip IV subfamily. Furthermore, we demonstrated that a cis-element identified using HD-Zip IV TFs from Arabidopsis is recognized by TFs from the phylogenetically distinct species wheat. The cDNAs isolated in the Y1H screen contained full-length coding regions because of the close position of DNA binding domains in HD-Zip IV TFs to the N-terminus of the protein. Therefore, the Y1H screen provides an efficient method to quickly isolate full-length sequences of very long and relatively low-abundant cDNAs from plants with yet unsequenced genomes. However, a prerequisite for success is a cDNA library with high sequence complexity, which is enriched with long cDNAs (Lopato et al., 2006).
The phylogenetic tree constructed using protein sequences of class IV HD-Zip TFs from different species revealed that GL9 proteins are relatively divergent from most other members of the family (Figure 2a). The closest known homologue of the wheat GL9 proteins is a HD-Zip IV transcription factor from rice, OsTF1 (Yang et al., 2002), which shares about 46% protein sequence identity with each of the GL9 TFs (Figure 2b). The expression pattern of OsTF1 has very little in common with the pattern of TaGL9 (Figure 3) and TdGL9H1 promoter activity in transgenic wheat (Figures 4 and 5). OsTF1 was isolated from young rice panicles; Northern blot analysis revealed its expression in the roots of seedlings and in the whole grain between 3 and 7 DAP (Yang et al., 2002). In situ hybridization showed strong OsTF1 gene expression throughout the embryo with strongest expression in the outer cell layer and, by 6 DAP, expression was found in the integument, the scutellar epithelium and the embryonic axis (Yang et al., 2002). Hybridization signals diminished after 6 DAP, coinciding with the commencement of endosperm starch filling, and after 10 DAP had totally disappeared. Expression of OsTF1 in the endosperm was detected at the same time as in the embryo; it was dispersed everywhere in the endosperm and disappeared together with the expression signals from embryo (Yang et al., 2002). Similar to OsTF1, TaGL9 expression started at 4 DAP, reached a maximum at 6 DAP and began to decrease at 7 DAP. However, expression of the wheat GL9 genes again increased at 8 DAP and was detected by Q-PCR until at least 20 DAP (Figure 3b). The Q-PCR data correlate with the results obtained from the analysis of GUS expression driven by the TdGL9H1 promoter, with the exception that strong expression of TaGL9 was found in the endosperm at 22 DAP (Figure 3a). However, activity of the TdGL9H1 promoter in the endosperm at the same stage was not detected (Figure 4c). In all three transgenic plant species, GUS activity was initially detected in the embryo and the area of the cellularising endosperm surrounding the embryo at 5 DAP (Figure 5a–d). It disappeared from the endosperm at 8–10 DAP but remained in the main vascular bundle of the scutellum until the end of grain desiccation and was still detectable in the harvested grain (Figure 4g–i). Hence, apart from sequence similarity of OsTF1 and wheat GL9 proteins, substantial differences were found in the expression of their genes.
No rice genes with a higher level of sequence identity to GL9 genes other than OsTF1 were found in rice sequence databases. Hence, it is possible that TaGL9 and TdGL9H1 are genes specific for Tritiaceae, and this could explain some of the differences seen in TdGL9H1 promoter activity in wheat and rice. We could not find a barley homologue of TaGL9 in public databases; however, because of overall similarity in gene sequences of wheat and barley, one can expect higher similarity of their promoter sequences and hence, higher similarity in spatial and temporal expression of GL9 genes from these plants. This expectation was confirmed in our experiments by detection of identical patterns of TdGL9H1 promoter activation in wheat and barley (Figures 4–6).
The pattern of TdGL9H1 expression suggests the involvement of this gene in the formation and/or maintenance of the main vascular bundle of the scutellum before imbibition and germination. The main vascular bundle of the scutellum has a critical role in controlling the mobilization and transport of endosperm reserves during germination (Price and King, 1973; Sopanen et al., 1977; Moshe Negbi, 1984). Because the activity of the TdGL9H1 promoter was rapidly lost during imbibition, it can be concluded that the gene has no role in vascular bundle regulatory function during germination. However, the possibility remains that TdGL9H1 is acting as a repressor of gene activity and during grain development keeping on hold some genes until imbibition and germination. These can be, for example, genes encoding sucrose transporters similar to the OsSUT1, which was demonstrated to be strongly induced in the scutellar vascular bundle of rice grains with 3 days post-imbibition but was not active in this tissue during grain development (Scofield et al., 2007). However, no known repressor motifs were identified in the GL9 proteins. Furthermore, in the Y2H assay, TaGL9 and TdGL9H2 proteins demonstrated the presence of an activation domain, which was roughly mapped to the N-terminal fragment of TaGL9 containing DNA binding and dimerisation domains (data not shown). These results suggest that the GL9 TFs function as activators rather than repressors and may have a role in the activation of genes involved in the development and maintenance of the vascular bundle before germination.
Only three genes have been reported for cereals to be specifically expressed in the main vascular bundle of the scutellum during grain development. They encode non-specific lipid transfer proteins (LTPs) from wheat: TaLtp7.2a, TaLtp9.1a and TaLtp9.3e (Boutrot et al., 2007). Promoters of these genes were fused to the uidA gene and their activity was studied in stably transformed rice plants. GUS activity was localized in the main vascular bundle of the scutellum (Boutrot et al., 2007). Because the spatial pattern of expression of these LTP genes coincided with that of the embryo provascular tissue development in rice and wheat (4–10 DAP) (Swift and O’Brien, 1970; Hoshikawa, 1989), it was speculated that these three LTPs may be involved in vascular tissue formation (Boutrot et al., 2007). Three other LTP genes reported on in the same paper, TaLtp7.1a, TaLtp9.2d and TaLtp9.4a, were expressed mainly in the epidermal cells of the embryo, a tissue that is covered by a cuticular layer. Roles in the secretion and/or deposition of extracellular lipophilic molecules on the protoderm layer during cuticle formation were assigned to these three LTPs (Boutrot et al., 2007).
We have found that the TdGL9H1 promoter was initially active in the epidermal cell layer of the embryo and in surrounding endosperm at the time of cuticle formation and later also detected in the walls of the main vascular bundle of the scutellum. These data may suggest that TdGL9H can be involved in both cuticle formation and development of a vascular bundle through the direct or indirect regulation of expression of the respective LTP genes with similar patterns of expression. However, such involvement remains to be confirmed by generation and analysis of transgenic wheat plants transformed with RNAi constructs fused to TdGL9H1 promoter or using other sophisticated approaches.
Furthermore, downstream genes regulated by the OUTER CELL LAYER1 (OCL1) gene of maize were recently discovered by a comparison of transcriptomes of transgenic maize plants over-expressing OCL1 and wild-type plants (Javelle et al., 2010). Analysis of annotations for 14 direct or indirect target genes showed that half of them encode proteins that are known to be involved in either biosynthesis or transport of lipids. One nonspecific LTP gene was also in the list of these proteins. All these data may suggest that GL9 TFs, which themselves contain a putative lipid-binding domain, might be involved in regulation of lipid biosynthesis and transport, initially in areas of cuticle formation between embryo and endosperm and later in the scutellar vascular bundle, although such involvement remains to be established.
TdGL9H1 promoter activity was not detected in the endosperm after 6 DAP, coinciding with endosperm starch filling. Diminishing and total vanishing of the GUS activity in the vascular bundle of the scutellum took place during seed imbibition and germination, when sugars start to move via the scutellar vascular bundle to the germinating embryo. Thus, promoter activity may be negatively regulated by high sugar concentrations. Such a scenario correlates with our prediction of several sugar-mediated repressor cis-elements in the TdGL9H1 promoter.
The promoter of TdGL9H1 is tissue specific and has relatively high transcriptional activity. It could thus be used for the engineering of cereal grain with altered scutellar vascular bundle properties with the aim of improving the efficacy of its function during the endosperm-dependent stage of seedling development and, thus, enhance the vigour of transgenic seedlings. Another possible biotechnological application for this promoter may be for specific expression of PR and R genes in the vascular bundle with the aim of protecting the dormant embryo from pathogen infection during seed storage. The TdGL9H1 promoter could also be used as a marker of embryonic vascular tissue formation during microspore embryogenesis, which is useful for the production of doubled haploid plants.
Gene cloning and plasmid construction
The full-length cDNA of TaGL9 was isolated from a yeast 2-hybrid cDNA library that was prepared from wheat grain at 0–6 DAP using the sequence CATTAAATG as bait, repeated four times in tandem, according to the procedure described by Lopato et al. (2006). The 3′ UTR of the cDNA sequence of TaGL9 was used as a probe to screen the BAC library prepared from the genomic DNA of T. durum cv. Langdon (Cenci et al., 2003) using Southern blot hybridization as described elsewhere (Sambrook and Russell, 2001). Five BAC clones (#1037 G19; #1078 B10; #1152 A6; #1286 C15 and #1129 D24) were identified; three of them (#1037 G19; #1078 B10 and #1286 C15) were selected for further analysis on the basis of the strength of the hybridization signals. BAC DNA was isolated and used as a template for PCR with primers derived from the coding region and the 3′ UTR of TaGL9. One BAC clone (#1037 G19) gave a PCR product. DNA isolated from the BAC (#1037 G19) was sequenced using the 454 sequencing method. The obtained gene sequence was subsequently used to design forward and reverse primers for the isolation of the promoter segment. The promoter with the full-length 5′-untranslated region was amplified by PCR using AccuPrime™Pfx DNA polymerase (Invitrogen, Mulgrave, Victoria, Australia) from DNA of BAC clone #1037 G19 as a template. It was cloned into the pENTR-D-TOPO vector (Invitrogen); the cloned insert was verified by sequencing and subcloned into the pMDC164 vector (Curtis and Grossniklaus, 2003) using recombination cloning. The resulting binary vector, designated pTdGL9H1, was introduced into Agrobacterium tumefaciens AGL1 strain by electroporation. For wheat transformation, the pTdGL9H1 construct was linearized using the unique PmeI site in the vector sequence.
The coding region of TdGL9H2 was isolated by nested RT-PCR using two sets of primers designed from the genomic sequence of TdGL9H1, and a mixture of RNA samples from developing grain (5–20 DAP) of T. durum as template. However, the expected coding region of TdGL9H1 was not isolated using this approach. The CACC sequence required for the directional cloning into pENTR-D-TOPO vector was introduced into the nested forward primer. For the Y1H assay, coding regions of TaGL9 and TdGL9H2 were amplified using forward and reverse primers with EcoRI and BamHI restriction endonuclease sites for TaGL9 and SfiI and SmaI for TdGL9H2, respectively. The amplified fragments were ligated into the respective restriction sites of the pGADT7 vector (Invitrogen).
For co-bombardment experiments, a 1378-bp-long promoter fragment of TaLtp9.1a was isolated using nested PCR and genomic DNA of T. aestivum cv Chinese Spring as a template. Primers were derived from the sequence of TaLtp9.1a gene (Acc. AJ852536). The CACC sequence introduced in the nested forward primer permitted directional cloning of the isolated PCR product into the pENTR-D-TOPO vector; the cloned insert was verified by sequencing and re-cloned into the pMDC164 vector. The resulting promoter-GUS fusion construct was designated pTaLtp9.1a-GUS. Full-length cDNAs of TaGL9 and TdGL9H2 were cloned into the pUbi vector (Morran et al., 2011), producing pUbi-TaGL9 and pUbi-TdGL9H2 constructs.
Plant transformation and analyses
The construct pTdGL9H1 was transformed into rice (Oryza sativa L. ssp. Japonica cv. Nipponbare) using Agrobacterium-mediated transformation and the method developed by Tingay et al. (1997) and modified by Matthews et al. (2001). Wheat (T. aestivum L. cv. Bobwhite) was transformed using biolistic bombardment as described by Kovalchuk et al. (2009). Transgene integration was confirmed by PCR using GUS-specific primers.
Whole-mount and histological GUS assays were performed as described by Li et al. (2008) using T0–T1 transgenic plants and T1–T2 seeds, respectively.
Q-PCR analysis of the expression of TaGL9 and TdGL9H1 genes in different tissues of wild-type wheat and at different stages of grain development was performed as described by Morran et al. (2011). Because of the absence of 3′ UTR region for TdGL9H2, specific primers could not be designed, and Q-PCR analysis of the TdGL9H2 gene expression was not performed.
We thank Anita Lapina for technical assistance with BAC DNA isolation and promoter cloning, Ursula Langridge, Lorraine Carruthers and Alex Kovalchuk for assistance with growing plants in the glasshouse and John Harris for critical reading of the manuscript. This work was supported by the Australian Research Council, the Grains Research and Development Corporation and the Government of South Australia.