title: Studies of a Brassica napus gene encoding a putative lipid transfer protein
Creators: name: Sohal, Awinder K.
Publisher: name: University of Glasgow
Year: 1997
Subjects: subject: Plant sciences.

description: The epidermis is an extremely important plant tissue because it is at the interface of the plant with its environment. To isolate genes expressed in the epidermis, a cDNA library constructed from leaves of Brassica napus was screened with an unidentified B. napus epidermis-specific, partial cDNA clone, pLFSA, which was isolated previously. Several full length cDNA clones were isolated, one of which was fully sequenced. This clone, designated BnLTP, encoded a putative non-specific lipid transfer protein (LTP) of 118 amino acids. Two other closely related cDNA clones, p2A4 and p5A9, were isolated and sequenced partially at the 3' ends. Sequence comparison of the three clones with pLF3A indicated the presence of four closely related but non-identical cDNA species. Based on this observation and previous Southern blot analysis of B. napus genomic DNA, it was concluded that BnLTP, p2A4, p5A9 and pLF3A were four members of a small closely related multigene family encoding putative LTPs. A genomic clone, designated []3.2A, homologous to BnLTP was isolated. A 4.8 kb Eco RI fragment from []3.2A, which hybridised strongly to the cDNA, was subcloned and sequenced. The fragment contained the full length gene, designated BnLTP. BnLTP encoded a 118 amino acid putative LTP, which differed at five amino acids from the cDNA. It consisted of two exons of 116 and 2 amino acids, respectively, interrupted by a 269 bp intron. The gene contained a 2.3 kb 5' upstream region that contained a TATA box and several cis-acting elements conserved in other LTP genes and genes involved in phenylpropanoid biosynthesis. B. napus LTP transcripts exhibited an organ-specific pattern of expression. They were expressed at high levels in leaves, stems, and floral tissues but were not detected in roots. In addition, endogenous LTP transcripts were induced in response to high white, blue and red light in B. napus and Arabidopsis leaves but not in response to UV-B light treatment. The BnLTP promoter was regulated in a spatial and temporal manner during development, as demonstrated by histochemical localisation of []-glucuronidase (GUS) in transgenic Arabidopsis plants carrying a 2.3 kb BnLTP promoter-GUS fusion (BnLTP-GUS). GUS was expressed at higher levels in younger developing leaves compared to older leaves. Cross-sections of transgenic leaf and stem tissue indicated that BnLTP-GUS was expressed predominantly in the epidermal cells. GUS activity was observed in the trichomes, epidermal pavement cells and guard cells. However, expression was also observed in the vascular bundles (xylem and phloem of leaves) and in the lateral root initials. In floral tissue, GUS was localised in sepals, stigmas, petals and stamens, but as the flower matured, it was expressed only at low levels in the stigma and sepal/petal abscission zone. However, GUS was not detected in mature petals but persisted in the stamens (pollen sacs and the filament). Expression of BnLTP-GUS in transgenic Arabidopsis was moderately induced in response to high white, blue and red light but not following UV-B light treatment of leaves. BnLTP-GUS was moderately induced in cold treated leaves and upon infection with CaMV but not in response to wounding. LTP expression increased in transgenic Arabidopsis plants constitutively expressing the CaMV Gene VI, which is essential for viral replication and disease symptoms. In Ar-abidopsis mutants altered in trichome development, glabrous 1 (gl1) and 2(gl2) and transparent testa glabra (ttg), endogenous LTP expression was not altered, and in each case expression was induced in response to high white light similar to wild type plants. However, in the photoregulation mutant icx1 (increased chalcone synthase expression), which is characterised not only by enhanced light stimulation of the epidermis-specific gene, CHS, but also by altered epidermal development, endogenous LTP expression was increased. In the double mutant ttg/icx1, which has phenotypic characteristics of both parents, endogenous LTP expression was similar to icx1.