The gene for the major cuticular wax‐associated protein and three homologous genes from broccoli (Brassica oleracea) and their expression patterns

Abstract

The gene for the major protein (WAX9) found in surface wax of broccoli, designated wax9D, and three homologous genes (wax9A, B and C) were isolated from a genomic library using the previously isolated cDNA encoding the WAX9 protein as the probe; all four genes were sequenced. Genomic Southern blot analysis using the WAX9 cDNA as a probe showed the presence of at least four homologous genes in broccoli genome. The sequence of the originally isolated WAX9 cDNA matched with that of gene D. All four genes have an intron two codons before the stop codon. The putative promoter regions of the four genes, beyond the first 200 bp immediately 5' to the translation start sites, are quite different. Essential elements such as TATA and CAAT boxes and several regions homologous to the promoter regions of other plant Itp genes were identified. The expression patterns of the genes were determined by RT-PCR with gene-specific primers and sequencing of the PCR products. All the genes were expressed in leaves and flower buds. While genes A, B and D also were expressed in stems and open flowers, expression of gene C was not detected in these organs. None of them were expressed in roots. The 972 bp 5'-flanking region of wax9D when fused to beta-glucuronidase (GUS) gene, directed tissue-specific GUS expression in transgenic tobacco plants; GUS expression was found in the epidermis of leaves, stems and flower petals, sepals, trichomes, and ovules but not in roots.

Tissue-Specific Expression and Promoter Analysis of the Tobacco ltp1 Gene

“…The observation of expression of the 1346-GUS construct in young cells of the germinating root led us to examine further the expression pattern in the germinating seedling, since both our previous data (Fleming et al, 1992) and those of other groups (Skriver et al, 1992;Kotilainen et al, 1994;Pyee and Kolattukudy, 1995) suggested that LTP gene expression is restricted to aerial portions of the plant. However, examples in which an LTP protein (Thoma et al, 1993) or ltp transcripts (Molina and García-Olmedo, 1993; Krause et al, 1994) are found in roots are known.…”

“…In situ hybridizations have shown accumulation of Itp transcripts in epidermal layers of tobacco (Fleming et al, 1992), tomato (Fleming et al, 1993), and Arabidopsis (Thoma et al, 1994), and anti-LTP antibodies recognized epitopes in cell walls and intercellular spaces in Arabidopsis (Thoma et al, 1993). Furthermore, in broccoli, LTP is the most abundant protein in the extracellular wax (Pyee andKolattukudy, 1994, 1995). Such a specific localization of LTP in the epidermal cell wall is incompatible with a role in general lipid redistribution, and, in the light of a11 these observations, LTPs have been proposed to play a role in the transport of extracellular lipophilic material.…”

Disruption of Glycosylphosphatidylinositol-Anchored Lipid Transfer Protein Gene Altered Cuticular Lipid Composition, Increased Plastoglobules, and Enhanced Susceptibility to Infection by the Fungal Pathogen Alternaria brassicicola

“…Plant LTPs are small (7-10 kD), abundant, and basic proteins that have a hydrophobic pocket capable of accommodating fatty acids or lysophospholipid molecules (Shin et al, 1995;Beisson et al, 2003). The results of plant genome and EST projects have demonstrated the presence of multiple LTP isoforms, which are speculated to be associated with diverse functions including cutin and wax assembly, pathogen defense, antifreezing, long-distance signaling, and cell wall loosening (Pyee and Kolattukudy, 1995;Molina and García-Olmedo, 1997;Maldonado et al, 2002;Beisson et al, 2003;Jeroen et al, 2005;Cameron et al, 2006;Roy-Barman et al, 2006;Choi et al, 2008). Most plant LTPs localize to the cell wall, which suggests that LTPs play a role in cutin monomers and wax transport (Pyee and Kolattukudy, 1995).…”

“…The results of plant genome and EST projects have demonstrated the presence of multiple LTP isoforms, which are speculated to be associated with diverse functions including cutin and wax assembly, pathogen defense, antifreezing, long-distance signaling, and cell wall loosening (Pyee and Kolattukudy, 1995;Molina and García-Olmedo, 1997;Maldonado et al, 2002;Beisson et al, 2003;Jeroen et al, 2005;Cameron et al, 2006;Roy-Barman et al, 2006;Choi et al, 2008). Most plant LTPs localize to the cell wall, which suggests that LTPs play a role in cutin monomers and wax transport (Pyee and Kolattukudy, 1995). The accumulation of cuticular wax on the leaves of tree tobacco (Nicotiana glauca) and the concomitant increase in LTP expression in response to drought stress indirectly suggest that LTP is involved in the accumulation of wax (Cameron et al, 2006).…”

Cryoprotectin: a plant lipid–transfer protein homologue that stabilizes membranes during freezing

“…Although this purified fraction showed only one band in a silver-stained SDS-PAGE gel, it contained more than one homologous polypeptide, as revealed by protein sequencing (Hincha et al 2001). All peptides belong to the family of WAX9 proteins (Pyee et al 1994;Pyee & Kolattukudy 1995), which show a high degree of sequence homology to plant LTPs (figure 4). WAX9 E, purified from the wax layer of cabbage leaves, has lipid transfer activity between liposomal membranes but no cryoprotective activity.…”