Cloning and expression analysis of candidate genes involved in wax deposition along the growing barley (Hordeum vulgare) leaf

Abstract

The aim of the present study was to isolate clones of genes which are likely to be involved in wax deposition on barley leaves. Of particular interest were those genes which encode proteins that take part in the synthesis and further modification of very long chain fatty acids (VLCFAs), the precursors of waxes. Previously, it had been shown that wax deposition commences within a spatially well-defined developmental zone along the growing barley leaf (Richardson et al. in Planta 222:472-483, 2005). In the present study, a barley microarray approach was used to screen for candidate contig-sequences (www.barleybase.org) that are expressed particularly in those leaf zones where wax deposition occurs and which are expressed specifically within the epidermis, the site of wax synthesis. Candidate contigs were used to screen an established in-house cDNA library of barley. Six full-length coding sequences clones were isolated. Based on sequence homologies, three clones were related to Arabidopsis CER6/CUT1, and these clones were termed HvCUT1;1, HvCUT1;2 and HvCUT1;3. A fourth clone, which was related to Arabidopsis Fiddlehead (FDH), was termed HvFDH1;1. These clones are likely to be involved in synthesis of VLCFAs. A fifth and sixth clone were related to Arabidopsis CER1, and were termed HvCER1;1 and HvCER1;2. These clones are likely to be involved in the decarbonylation pathway of VLCFAs. Semi-quantitative RT-PCR confirmed microarray expression data. In addition, expression analyses at 10-mm resolution along the blade suggest that HvCUT1;1 (and possibly HvCUT1;2) and HvCER1;1 are involved in commencement of wax deposition during barley leaf epidermal cell development.

Evolutionary Conserved Function of Barley and Arabidopsis 3-KETOACYL-CoA SYNTHASES in Providing Wax Signals for Germination of Powdery Mildew Fungi

“…Based on BLAST analysis, Li et al (2013) suggested barley EST DQ646644 (named in this study HvCER6 and identical to HvCut1.3; Richardson et al, 2007) as a potential homolog for At1g68530. In their study, Li et al (2013) placed 27 barley cer loci, none of which have been cloned so far, on a barley consensus map.…”

“…sequence corresponds to the barley high-confidence gene MLOC_51583 that was previously referred to as HvCUT1.3 (Richardson et al, 2007). Sequencing of a PCR product amplified from genomic DNA from Ingridmlo5 or lwa1 plants with primers specific for the LWA1 gene identified a single-nucleotide acid polymorphism in the mutant genotype, which led to an amino acid change in the deduced protein at position L 136 F (Supplemental Fig.…”

“…In Arabidopsis, this gap was closed by reverse genetic approaches, which enabled the identification of additional genes involved in cuticular wax biosynthesis (Kunst and Samuels, 2003). Sequence homologies to some of these genes were used to clone candidate genes of barley (Richardson et al, 2007). However, because forward and reverse genetic data are missing, gene-function relationships in barley are still speculative.…”

Cell-Specific Vacuolar Calcium Storage Mediated by CAX1 Regulates Apoplastic Calcium Concentration, Gas Exchange, and Plant Productivity in Arabidopsis

“…All three transcripts were more highly expressed in the mesophyll; however, PHT4.4 has previously been localized to the chloroplast (Roth et al, 2004), and PHO1-H8 and KEA1 are predicted to be targeted to the mitochondria and chloroplast, respectively (SUBA database) (Heazlewood et al, 2007). Further support for the role of KEA1 in chloroplasts comes from its closest two homologs in barley also being highly expressed in the mesophyll (Richardson et al, 2007). Therefore, none of these candidates are likely to encode a transporter that directly facilitates epidermal enrichment of vacuolar [P] or [K].…”

cDNA cloning and expression analysis of a putative decarbonylase TaCer1 from wheat (Triticum aestivum L.)

“…The promoter of Cer1 is of tissue specificity, expressing in epidermis and young tissue (Karaba 2007). The function of Cer1 is also testified by Richardson et al (2007). They isolated HvCer1 from barley and speculated that it was likely to be involved in the decarbonylation pathway of VLCFAs.…”