| We isolated a laccase cDNA, GaLAC1, from the library of Gossypium arboreum L. suspension cells. Sequence analysis showed that the GaLAC1 cDNA contained a open reading frame coding for a protein of 566 amino acid residues (63.74KD), with an N-terminal signal peptide of 23 amino acids, four Cu2+ binding domains, and 17 putative carbohydrate moieties. BLASTP showed that GaLAC1 is most similar to a tobacco laccase with a sequence identify of 45%.Northern blot and RT-PCR analysis indicated GaLAC1 was expressed in cotton roots, and in aerial tissues the GaLAC1 transcripts were undetectable. The transcript level in roots of a glandless cultivar (G. hirsutum cv. hai-1) was greatly reduced in comparison with glanded cotton cultivar. However, the transcription was significantly induced in roots of this glandless cultivar upon elicitation by the Verticilium dahlie elicitor. The expression pattern of GaLAC1 in roots was similar to genes involved in gossypol biosynthesis pathway. The immunolocalization of GaLAC1 and histochemical staining of gossypol experiments revealed that both laccase and gossypol were located in outer layer-cells of the root tip, this suggests that GaLAC1 could be involved in crosslinking of gossypol to the cell wall. Southern blot revealed that several members of GaLAC1 were present in the diploid G. arboreum genome. In Arabidopsis plants transformed with GaLAC1promoter::GUS construct, the GUS activity was detected solely in root tip, consistent with the expression pattern of GaLAC1 in cotton.To investigate biochemical properties of GaLAC1 in vitro, two distinct expression vectors, pPIC3K/GaLAC1 and pPIC9K/GaLAC1, were constructed. In pPIC3K/GaLAC1 the complete open reading frame of GaLAC1 was inserted, including the native signal sequence of GaLAC1. In pPIC9K/GaLAC1 the signal sequence of GaLAC1 was substituted by the vector signal for target protein secretion. For both expression vectors, the laccase activity was found in the supernatant of the culture medium, indicating that GaLAC1 was secreted from Pichia cells, and the signal peptide of GALAC1 also has secretory activity in Pichia. The crude enzyme preparation of the culture medium showed clear oxidization activities toward a synthesized substrate, ABTS. The activity was also high with sinapinic acid and sinapic alcohol as substrates, but low with syringaldazine, syringic acid, ferulic acid and vanillic acid. No activity was detected when incubated with catechol. The inhibitory effects of metal chelators were examined during oxidation of ABTS. Copper ion chelators such as L-cysteine, EDTA, NaF, SDS inhibited the enzyme activity to different extents. The optimal activity for oxidation of ABTS was obtained at pH 4.0.In order to discover the biological function of GaLAC1, a chimeric CaMV35S::GaLAC1 gene was introduced into Arabidopsis thaliana via Agrobacterium-mediated transformation. A number of transgenic plants showed obvious laccase activity and GaLAC1 expression. We then selected five transgenic lines for further analysis. In normal growth conditions, the transgenic plants showed no visible phenotypic changes. Methanol-soluble phenolic compounds were quantified based on their reactivity to Folin's regent, the transgenic seedlings exhibited only slightly decrease in soluble phenolic content. The phenolic compounds were then separated by HPLC, and six compounds were identified in combination with Mass Spectra: 1, sinapoyl glucose; 2, Rha-Glc-Rha-kaempferol; 3, Glu-Rha-kaempferol; 4, Rha-Rha-quercetin; 5, sinapoyl malate; 6, Rha-Rha-kaempferol. While the flavonoid profile in transgenic (LAC 4-2) and WT seedlings was similar, the accumulation of sinapic esters was clearly changed in that the peak 5 (sinapoyl malate) was greatly reduced in LAC 4-2 seedlings. Among the cell wall-bound phenolics washed by alkaline, transgenic seedlings (LAC 4-2) contained a higher amount of trans-ferulic acid than the WT. When the four-day-old seedlings with about 1-cm-long roots were transferred into a second agar medium supplemented with d...
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